XX Annual Meeting of the International Society for Heart Research Latin American Section

Journal of Molecular and Cellular Cardiology 53 (2012) S1–S17

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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y j m c c

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XX Annual Meeting of the International Society for Heart Research Latin American Section Dr. Lorenzo Sazie Lecture Hall, School of Medicine, University of Chile, Santiago, Chile, October 25–26, 2012 Differences in the signaling pathways involved in physiological and pathological cardiac hypertrophy development: Putative targets for novel therapeutic strategies I.L. Ennis Centro de Investigaciones Cardiovasculares, Fac. de Cs. Medicas, UNLPCONICET, Argentina Sustained hemodynamic overload induces the development of cardiac hypertrophy (CH). It can be distinguished between physiological CH, an adaptive response to an increase in workload induced by physical training or pregnancy, and pathological CH that is related to cardiovascular pathologies such as hypertension or cardiac infarction and represents an independent risk factor for cardiovascular morbidity and mortality. The hyperactivity of the cardiac Na+/H+ exchanger (NHE1) has been widely demonstrated to play a key role in pathological CH by augmenting intracellular Na+ content, favoring the reverse mode of the Na+/Ca2 + exchanger increasing intracellular Ca2 + and activating the calcineurin/NFAT signaling pathway. Calcineurin, a phosphatase activated by increases in intracellular Ca2 +, is critical for pathological but not physiological CH. The NHE1 has been shown to be hyperactive not only in several experimental models of CH but also in failing human myocardium. Likewise, NHE1 specific inhibition prevents or induces the regression of pathological CH while its hyperactivity is sufficient to induce CH development. However, the role of NHE1 in physiological CH has not been elucidated yet. We hypothesize that the prevention of its activation, possibly by an inhibitory AKT-dependent phosphorylation of the cytosolic tail could potentially drive CH development to the physiological pattern instead to the pathological one under hemodynamic overload.

Rho kinase activation and cardiovascular remodeling J.E. Jalil División de Enfermedades Cardiovasculares, Escuela de Medicina, PUC, Santiago Chile One novel intracellular mechanism that triggers remodeling is the small guanosine triphosphatase Rho and its target Rho kinase (ROCK). Both have important roles in blood pressure regulation, vascular 0022-2828/$ – see front matter © 2012 Published by Elsevier Ltd. doi:10.1016/j.yjmcc.2012.07.007

smooth muscle contraction, and cardiovascular (CV) and renal remodeling. Rho is activated by agonists of receptors coupled to the cell membrane G protein, such as angiotensin II and noradrenaline. Activated ROCK plays a significant role in vascular smooth muscle cell contraction and regulates several cellular functions (such as actin cytoskeleton organization, adhesion and motility, proliferation, differentiation, apoptosis and cell survival). ROCK also mediates upregulation of several proinflammatory, thrombogenic and fibrogenic molecules and downregulation of endothelial nitric oxide synthase. Recently, progressive ROCK activation in circulating leukocytes in patients with CV disease has been observed, which is directly related to cardiac remodeling and LV dysfunction. Highest ROCK activation was observed in HF patients. Thus, ROCK activation in circulating human leukocytes is related to both pathological CV remodeling and risk in a direct way. ROCK activation is a key signaling pathway of progressive organ damage and a possible pathological molecular biomarker of CV remodeling and inflammation which is mirrored in circulating leukocytes. Its pathological significance, activation mechanisms, and consequences during pathological CV remodeling need further investigation. Fondecyt 1121060

Interaction of endoplasmic reticulum and mitochondria induced by GLP-1 in vascular smooth muscle cells M. Chiong, P.E. Morales, G. Torres, L. Michea, S. Lavandero Center Molecular Studies of the Cell, Faculties of Chemical and Pharmaceutical Sciences and Medicine, University of Chile Mitochondrial function can be regulated by the endoplasmic reticulum (ER) through a functional coupling between these two organelles. Upon a stimulus, mitochondria moves towards the perinuclear region and physical contacts between ER and mitochondria can be observed. Ca2 + is released from the ER through IP3 receptor and enters the mitochondria though low affinity Ca2 + channels. Transient mitochondrial Ca2 + rise activates Kreb cycle dehydrogenases and both oxygen consumption and ATP level increase is observed. However, long and high mitochondrial Ca2 +

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rise induces cytochrome c release and cell death by apoptosis is triggered. Mitochondrial function is very important in cardiac myocytes and vascular smooth muscle cells (VSMC), regulating both phenotype and function. Moreover, several hormones, including the incretin GLP-1, can modulate mitochondria-ER coupling suggesting a new mechanism for hormonal cardiovascular function regulation. Fondecyt 1110180 (MC and LM) and Anillo ACT1111 (MC and SL). PEM and GT hold Conicyt fellowships.

Reperfusion arrhythmias Leticia Vittone Centro de Investigaciones Cardiovasculares, CCT-La Plata CONICETUNLP, Argentina Reperfusion of the myocardium after a relatively brief period of ischemia may precipitate a pattern of arrhythmias with different levels of severity. This is important in the clinical setting as revealed by the numerous reports indicating a significant role for these arrhythmias either during spontaneous reperfusion after coronary spasm or thrombolysis subsequent to acute myocardial infarction. Earlier experimental work has revealed that the return to normal pH after a period of acidosis can evoke cardiac arrhythmias that are dependent on CaMKII. This pattern of arrhythmias was initiated by a focal triggered mechanism, involving spontaneous diastolic Ca2 + release from cardiac ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Because of acidosis is a major component of myocardial ischemia and CaMKII was found to be activated at the beginning of reperfusion, we explored the relationship between reperfusion arrhythmias and the CaMKII-phosphorylation of phospholamban and RyR2, two proteins critically involved in the SR Ca2 + handling. The relative contribution of these two CaMKII targets was studied in transgenic mouse models. Furthermore, since oxidative stress is another hallmark event upon reperfusion we also investigated the role of redox modifications of RyR2 (S-glutathionylation and S-nitrosylation) upon arrhythmias appearance. While CaMKII-phosphorylation of SR proteins seems to favour arrhythmias, the S-glutathionylation of RyR2 may partially prevent them.

Recruitment of sarcoplasmic reticulum function by inotropic stimulation: A mechanism of inotropic reserve in neonatal cardiomyocytes R.A. Bassani, J.W.M. Bassani CEB/Universidade Estadual de Campinas, Campinas, SP, Brazil The sarcoplasmic reticulum (SR) is the main source of Ca2 + for the excitation-contraction coupling process (ECC) in adult mammalian cardiomyocytes. However, in neonates, the organelle is still immature and is considered to contribute little to ECC. Accordingly, under basal conditions, the SR Ca2 + content ([Ca2 +]SR) in neonatal rat myocyes is 25% lower than in adults, and the fraction of this content released at a twitch (fractional SR Ca2 + release, FR) is only 0.35 (vs. 0.65 in adults). Maximal stimulation of neonatal cells with noradrenaline (NA) increases [Ca2 +]SR by 50% and elevates FR to 0.55. As a result, the contribution of SR Ca2 + release to ECC, estimated as 65% in unstimulated cells, may reach as much as 80%. Moreover, complete inhibition of the SR function suppresses the maximally NA-stimulated total Ca2 + flux by ~ 90%. It thus may be concluded that recruitment of SR function by adrenergic stimulation markedly enhances the participation of SR Ca2 + release in ECC, which seems to constitute an important mechanism of inotropic reserve in the neonate, and is

crucial for effective adrenergic increase of Ca2 + mobilization (support: FAPESP and FAEPEX/UNICAMP).

Intracellular Ca2 + dynamics in ischemia/reperfusion A. Mattiazzi Centro de Investigaciones Cardiovasculares, CCT-CONICET-La Plata, Facultad de Medicina, UNLP, Argentina We previously demonstrated the beneficial effect of calciumcalmodulin-dependent protein kinase (CaMKII) phosphorylation of sarcoplasmic reticulum (SR) proteins at the onset of reperfusion, after a relatively short ischemic period [stunned heart, (Am J Physiol., 2003)]. We recently showed however, that CaMKII-dependent phosphorylation of SR proteins is also detrimental, since it contributes to reperfusion arrhythmias (J Mol Cell Cardiol, 2011). Now, the critical question is: which are the mechanisms that link the increase in CaMKII-dependent phosphorylation of SR proteins with either a beneficial or detrimental effect in I/R? A necessary step to answer this paradox is to explore Ca2 + dynamics at the cytosol and SR during I/R. We used Pulsed Local Field Fluorescence microscopy, for simultaneous determination of SR and cytosolic Ca2 + and mechanical parameters, and confocal microscopy to study Ca2 +sparks and waves during I/R, in the intact heart. The results indicate that CaMKIIdependent phosphorylation of phospholamban at the onset of reperfusion is a main responsible for the recovery of intracellular Ca2 + transient. These findings support the beneficial effect of CaMKII on contractility during reperfusion. Moreover, the increase in CaMKIIdependent phosphorylation of RyR2 at the onset of reperfusion is associated with an abrupt increase in cytosolic Ca2 + released by the SR and an increase in Ca2 + waves. These events may constitute the underlying mechanism of the detrimental effect of CaMKII in I/R, i.e. the observed CaMKII-dependent reperfusion triggered arrhythmias.

Na + overload as a mediator of apoptosis and arrhythmias in heart failure: Role of CaMKII M. Vila Petroff Centro de Investigaciones Cardiovasculares, Facultad de Medicina UNLP, 60 y 120S/N La Plata, Argentina Heart failure (HF) is associated with enhanced incidence of arrhythmias and apoptosis, having both these processes been attributed, at least in part to alterations in intracellular Ca2 + handling. Given that Ca2 +i regulation is linked to Na+i homeostasis through its effect on Na+/Ca2 + exchanger (NCX) activity and that Na+i levels are increased in the failing heart, it is plausible that elevated Na+i is the initiating step that induces apoptosis and arrhythmias in HF. In addition, the calcium/calmodulin-dependent protein kinase II (CaMKII) is overexpressed in HF and has been shown to mediate apoptosis and arrhythmias. However, whether elevated Na+i leads to an increase in Ca2 +i that results in the activation of CaMKII which enables apoptotic and proarrhythmogenic activity has not been evaluated previously. In this study we examined whether elevated Na+i can promote apoptosis and arrhythmias through CaMKII-dependent signaling. For this purpose, Na+i was increased in rat cardiac myocytes by inhibiting the Na+/K+-ATPase (NKA) with ouabain. Na+i elevation enhanced CaMKII activity, induced apoptosis and promoted arrhythmias, effects that were blocked by CaMKII inhibition (KN93; AIP) and enhanced by CaMKII overexpression. Elevated Na+i failed to induce apoptosis and arrhythmias in myocytes isolated from transgenic mice expressing an SR-targeted CaMKII

Abstracts

inhibitory peptide (SR-AIP) and both effects were prevented by stabilizing the ryanodine receptor (RyR) with tratacaine and carvedilol. We conclude that elevated Na+i activates CaMKII, which through the phosphorylation of its SR targets promotes apoptosis and arrhythmias. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in SR Ca2 + leak, could be the common underlying mechanism involved.

Reactive oxygen species as second messengers involved in signaling pathways triggered by angiotensin II and aldosterone E.A. Aiello Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, UNLP-CONICET, La Plata, Argentina During many years the reactive oxygen species (ROS) were exclusively considered as deleterious agents. However, over the last few years, many evidences supported the idea that ROS can also act as second messengers, mediating several intracellular signalling pathways. Consistently, we proposed the participation of these oxidative agents as mediators of certain cardiac physiological effects of angiotensin II (Ang II) and aldosterone. These effects are produced by the stimulation of the so called mechanism “ROS-induced-ROS release”, by which mitochondrial ROS are released to the citosol after the activation of the mitochondrial ATP-dependent K+ channels by ROS produced by the NADPH oxidase. We have proposed that these mitochondrial ROS stimulates the MAP kinase ERK, which in turn activates the Na+/H+ exchanger (NHE-1), inducing an increase in intracellular Na+([Na+]i). This [Na+]i enhancement promotes the increase in intracellular Ca2 + ([Ca2 +]i) via the reverse mode of the Na+/Ca2 + exchanger (NCX), leading to a positive inotropic effect. This increase in [Ca2 +]i could also trigger the development of cardiac hypertrophy by the calcineurin pathway. In addition, we have recently demonstrated that mitochondrial ROS are also able to stimulate the Na+/HCO3− cotransport (NBC), a mechanism that, together with the NHE-1, regulates cardiac intracellular pH and [Na+] i by promoting the co-influx of Na+ and HCO3− into the cell.

Protein disulfide isomerase: An endoplasmic reticulum chaperone involved in redox signaling and homeostasis R.M. Francisco Laurindo Vascular Biology Laboratory, Heart Institute(Incor), University of São Paulo School of Medicine, Brazil Protein folding at the endoplasmic reticulum(ER) lumen involves chaperone-assisted folding, glycosylation and disulfide bond introduction, the later consisting in the transfer of oxidizing equivalents to cysteine thiols at their correct location in nascent proteins. The main effectors of disulfide bond introduction are protein disulfide isomerase(s), particularly PDIA1 (PDI). ER-based PDI is an abundantly expressed thioredoxin superfamily oxidoreductase displaying many interactions with redox and nonredox proteins and several posttranslational modifications. PDI family contains N20 members with some apparent complementary actions. PDI has oxidoreductase, isomerase and chaperone effects, the latter not directly dependent on its thiols. PDI is a converging hub for pathways of disulfide bond introduction into ER-processed proteins, via hydrogen peroxidegenerating mechanisms involving the ER flavooxidase Ero1α, as well as hydrogen peroxide-consuming reactions involving peroxiredoxin IV and novel peroxidases Gpx7/8. A situation in which ER-dependent reactive oxygen species (ROS) generation is increased in many cell

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types is ER stress. PDI is one candidate pathway for coupling ER stress to ROS generation. Emerging information suggests a convergence between PDI and Nox family NADPH oxidases. In vascular smooth muscle cells, PDI silencing prevents Nox responses to angiotensin-II and inhibits Akt phosphorylation in vascular cells. Also PDI is required for Nox1/ROS-dependent vascular smooth muscle cell migration through pathways involving small GTPases Rac1 and RhoA, while PDI silencing promotes cytoskeletal disruption. PDI overexpression spontaneously enhances Nox activation and expression. During acute ER stress in smooth muscle cells, silencing of either Nox4 or PDI abolishes ROS generation, while PDI silencing enhances apoptosis. During sustained ER stress, ROS generation correlates with increased apoptosis, but does not induce cell death. At the cell surface, PDI mediates redox-dependent adhesion, coagulation/thrombosis, immune functions and virus internalization. The route of PDI externalization remains elusive. Thus, such multiple effects render PDI(s) putative redox cell signaling adaptors of broader significance. Altogether, redox pathways associated with (patho)physiological protein folding are prime candidate regulators of cellular redox status in many diseases (Research supported by: FAPESP, CNPq/INCT Redoxoma).

Cardiac metabolism in heart failure P. Castroa, H. Verdejoa, J.L. Wintera, R. Altamiranoa, R. Melladob, J. Kuzmicicc, S. Lavanderoc, M. Chiongc a Clinical Hospital, Faculty of Medicine, P. Catholic University of Chile, Chile b Faculty of Chemistry, P. Catholic University of Chile, Chile c Center of Molecular Studies of the Cell, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Chile Heart Failure (HF) continues to be a major health problem. Although a better knowledge of its physiopathology and advances in treatment of this disease has significantly lowered its mortality, it still remains around 50% at 5 years. It is unknown why heart failure progresses even when patients are treated with the best therapy available. Evidences suggest that heart failure progression is due to loss of neurohumoral blockade in advanced stages of the disease and to alterations in myocardial metabolism induced, in part, by this neurohumoral activation. Alterations in cardiac energy metabolism, especially those related to substrate utilization and insulin resistance, reduce the efficiency of energy production, causing a heart energy reserve deficit. These events play a basic role in heart failure progression. Therefore, modulation of cardiac metabolism has arisen as a promissory therapy in the treatment of heart failure. A description of myocardial energy metabolism, the role of impaired energy metabolism in heart failure progression and new therapies for heart failure involving metabolic intervention will be discussed. Fondecyt 1090727 and Anillo ACT1111 (SL and MC).

Importance of phospho-GSK-3β in ischemic pre and postconditioning in SHR S. Mosca Centro de Investigaciones Cardiovasculares CCT-CONICET, Facultad de Cs Médicas, Universidad Nacional de La Plata, La Plata, Argentina P-GSK-3β mediates the cardioprotection achieved by ischemic pre (PRE)-and postconditioning (POS) in normotensive animals. Our aim was to determine the role played by that enzyme in spontaneously hypertensive rats (SHR). Isolated hearts were submitted to 45 min

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global ischemia (GI) and 1 h reperfusion (R). In other groups a cycle of 5 min GI and 10 min of R was applied prior to 45 min-GI (PRE) or three cycles of 30 s-GI/30 s-R was applied at the start of R (POS). Other hearts received 3 mM LiCl – a GSK-3β inhibitor – 10 min before GI or during the first 3 min of R. Infarct size (IS) was determined by TTC staining. The expression of P-GSK-3β and P-Akt in cytosolic (cyt) and mitochondrial (m) fractions and P-GSK-3β/VDAC physical associations were assessed. The reduced glutathione (GSH) content, activity and expression of MnSOD and Ca2 +-induced mPTP opening were also measured. IS was significantly diminished by PRE, POS and LiCl treatments. The cyt P-GSK-3β and P-Akt contents and physical associations decreased in IC hearts and were restored after interventions. The increases of cyt MnSOD activity and decreases of GSH content and mPTP opening of IC hearts were attenuated by the interventions. The results show that an attenuation of mPTP opening mediated by cyt P-GSK-3β/VDAC association is the main mechanism contributing to the reduction of IS and oxidative stress achieved by PRE and POS in SHR hearts.

Ischemia/reperfusion injury: Lessons from transgenic animals M. Salas Centro de Investigaciones Cardiovasculares, CCT-CONICET-La Plata, Facultad de Ciencias Médicas, UNLP, Argentina Myocardial infarction is the leading cause of morbimortality in the Western world. Considerable efforts are being done to elucidate the processes involved in the myocardial damage. The restoration of coronary flow is accompanied by reperfusion-induced cardiac dysfunction, cell damage or ventricular arrhythmias. Alterations in Ca2 + homeostasis and sarcoplasmic reticulum (SR) Ca2 + handling emerge as central contributors to post-ischemic dysfunction and injury. In previous work we showed that SR CaMKII-dependent phosphorylations as well as mitochondria dysfunction are involved in the mechanism of injury (Salas et al., 2010). In order to elucidate which of the CaMKII dependent SR phosphorylatable sites was responsible for the deleterious effect of the kinase we performed global I/R in isolated Langendorff perfused hearts of different transgenic mice strains: a) SR-AIP expressing inhibition of CaMKII targeted to the SR, b) DM having both phospholamban (PLN) phosphorylatable sites: Thr17 (CaMKII) and Ser16 (PKA) mutated to alanine, c) S2814A in which the CaMKII dependent site, Ser2814 of RyR2 was mutated to alanine. Age-matched wild type mice (WT) served as controls. Our results suggest that 1. CaMKII dependent PLN phosphorylation has a beneficial role in the post-infarct recovery; 2. CaMKII dependent RyR2 phosphorylation, is involved in the deleterious effect of CaMKII possibly by increasing diastolic Ca2 leak and mitochondrial Ca2 + overload.

Myocardial regeneration with genetically modified mesenchymal stromal cells A. Crottogini Favaloro University, Buenos Aires, Argentina In humans and other large mammals the adult cardiomyocyte is not a post-mitotic cell. In pigs with myocardial infarction, we have shown that a plasmid encoding human vascular endothelial growth factor (pVEGF) induces adult cardiomyocyte mitosis as well as recruitment and activation of cardiac progenitor cells. On the other hand, the paracrine effect of implanted stem cells has been claimed to be of cardinal relevance in tissue renewal. We are thus testing the

efficacy of bone marrow mesenchymal stromal cells (MSCs) genetically modified to overexpress human VEGF165 on vascular and muscle regeneration in large mammalian models of ischemic heart disease and rabbit models of peripheral artery disease. In sheep with acute myocardial infarction, the intramyocardial injection of allogeneic MSCs previously transfected with pVEGF induces a significant 33% reduction in infarct size, as compared with non-transfected MSCs and pVEGF alone. This effect is accompanied by a significant improvement in left ventricular (LV) function, as indicated by decreased LV remodeling, with end systolic volume and ejection fraction values achieving their baseline, pre-infarct, levels. The mechanisms involved have not yet been disclosed. However, these preliminary results suggest that allogeneic MSCs genetically modified to overexpress mitogenic growth factors may prove potentially useful in the clinical setting to limit infarct size and prevent, or even reverse, the deterioration of LV function.

Apoptosis or survival of cardiac fibroblasts and myofibroblasts and its relation to the development of cardiac fibrosis Guillermo Díaz-Araya Laboratorio de Farmacoquímica, Departamento de Química Farmacológica y Toxicológica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile In the heart cardiac fibroblasts (CF) is the most numerous cell type, upon TGF-β1 they are differentiated in myofibroblast (CMF), which secretes higher amounts of extracellular matrix proteins (ECM). Under physiological conditions CF are responsible for the ECM homeostasis in heart tissue; however, in conditions of tissue damage, the CMF are primarily responsible for the healing process. Diverse mechanisms modulate the survival or apoptosis of CF and CMF, which will directly regulate turnover of ECM proteins; and its excessive deposit leads to cardiac fibrosis. Normally CF and CMF express the angiotensin II subtype AT1 receptor, which induces proliferation and secretion of collagen type I. However, their overexpression induces apoptosis of CF greater extent than in CMF. In addition, CF and CMF express kinin B1 and B2 receptors. The activation of these receptors, by their respective agonists, reduces collagen secretion, without affecting cell viability. Similarly, in CF through beta-2 adrenergic receptor, isoproterenol triggers CF autophagy, decrease collagen synthesis and induce proliferation, whereas no effect was observed in CMF. Finally, simulated ischemia/ reperfusion, or simvastatin triggers CF and CMF apoptosis, although in greater magnitude in CF than in CMF. Conclusion: CMF have greater resistance to cellular death mechanisms that CF, and its excessive survival may contribute to the development of cardiac fibrosis.

Insulin and VCAM-1 protect cardiomyocytes from ischemia death C. Humeresa, A. Díaza, G. Corderoa, V. Gonzáleza, R. Corbalánb, L. Garcíaa a Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile b Facultad de Medicina, Universidad Católica de Chile, Chile Vascular cellular adhesion molecule-1 (VCAM-1) is a cell adhesion molecule of the immunoglobulin family involved in inflammatory processes. This protein is essential for other non-immune functions like heart development and homeostasis. In this regard mice null for this protein are not viable. Our data indicate that rat cardiomyocytes express VCAM-1 and that insulin increases its levels. Insulin exerts

Abstracts

cardioprotection by triggering anti-apoptotic and proliferative effects. Here we described whether insulin and VCAM-1 exert protective effect on cardiomyocyte under ischemia. Neonatal rat cardiomyocytes were stimulated with 10 and 100 nM insulin before or during simulated ischemia. At the end of 8 hours of ischemia we evaluated cell viability by Trypan blue exclusion, subG1 apoptotic population by flow cytometry, LDH release, and VCAM-1 by immunowestern blot. Effect on viability of α4β1 integrin (VCAM-1 receptor) stimulation on ischemia was assessed by TUNNEL. Incubation with 10 nM insulin during ischemia produced a significant reduction of both necrotic and apoptotic cell death and this was related to an increase in VCAM-1 expression. However, 100 nM did not protected against cell death. α4β4 integrin protected from cell death in the same extent than insulin. Insulin has cytoprotective effect and this effect is associated with increased VCAM-1. This work establishes a link between cellular interactions and the protection of cardiomyocytes. Fondecyt No. 1110346.

Impact of impaired glucose tolerance and insulin resistance in heart injury: Role of Ca2 +-calmodulin kinase II (CaMKII) L. Sommesea, P. Blancob, O. Velez Ruedaa, C. Castroa, C. Zanuzzib, E. Portianskyb, A. Mattiazzia, J. Palomequea a Fac. de Cs. Médicas, UNLP, CONICET-CCT La Plata, Argentina b Fac. de Cs. Veterinarias, UNLP, CONICET-CCT La Plata, Argentina Although impaired glucose tolerance (IGT) and CaMKII are linked to cardiac disease, the nexus between IGT and CaMKII in the heart has never been explored. To reach this goal we used an IGT model induced by a fructose-rich diet (control, C; and fructose, F; rats or mice), and we performed echocardiography, biochemical studies, + TBARS, and Cai2measurements. F rats showed decreased contractility and increased hypertrophy by echocardiography associated with increased CaMKII activity (P-CaMKII 100.2 ± 24.2% and P-Thr17 of phospholamban 169.0 ± 51.4%), P-p38MAPK (61.7 ± 13.9%), ROS (106.7 ± 28.5%) and apoptosis (increased Bax/Bcl2 [273.6 ± 39.7%] and TUNEL positive cells) with respect to C rats. Myocytes from F rats showed decreased inotropic reserve to isoproterenol and increased non stimulated events, which were prevented by pretreatment of the myocytes with KN93, a CaMKII inhibitor. ROS increased either in F mice which express a CaMKII inhibitor (AC3I) or in the control strain (AC3C), however, C AC3I showed lower ROS than C AC3C, indicating that ROS production is both, dependent and independent of CaMKII activity. Altogether, the results would indicate a link between CaMKII activation and TGA.

Testosterone increases GLUT4-dependent glucose uptake through CAMKII/AMPK in cardiomyocytes C. Wilson, A. Contreras, K. Montoya, M. Estrada ICBM, Facultad de Medicina, Universidad de Chile, Chile Metabolic changes are crucial adaptations during the development of cardiac hypertrophy, mainly to supply high contractile and growth demands under this condition. Testosterone induces cardiac hypertrophy; however, their metabolic effects in the heart are unknown. In this work, we study the effect of testosterone on glucose uptake through glucose transporter 4 (GLUT4) and the CaMKII/AMPK pathway in cardiomyocytes. The main findings from our work show that testosterone increased glucose uptake in rat neonatal cardiomyocytes, which was blocked by indinavir, a specific GLUT4 blocker. In addition, both

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glucose uptake and GLUT4 cell surface exposure induced by testosterone are mediate by activation of the CaMKII/AMPK pathway. These results suggest that testosterone increase glucose uptake through GLUT4 and CaMKII/AMPK pathway in cardiomyocytes. Glucose uptake through these intermediaries could represent a mechanism by which testosterone increases energy production for cell growth, hypertrophy or other anabolic processes in cardiomyocytes. FONDECYT 1120259

Kinin B1 receptor activation through PKC-PLA2-COX2 and PGI2 secretion reduces collagen I expression in rat neonate cardiac myofibroblast M. Catalána, P. Bozaa, I. Olmedoa, M.V. Velardeb, G. Díaz-Arayaa a Laboratorio de Farmacoquímica, Departamento de Química Farmacológica y Toxicológica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile b Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológícas, Pontificia Universidad Católica de Chile, Chile BK activates the B2 receptor in cardiac fibroblast (CF) reducing secretion of collagen. However, the presence and role of B1 receptor in collagen synthesis has not been characterized in cardiac myofibroblast (CMF). Primary cultures of CF in passage 2 were differentiated to CMF by TGF-b1. Collagen I synthesis was evaluated by western blot. The participation of PKC, PLA2 and COX2 was evaluated by specific inhibitors. The participation of PGI2 receptor was evaluated by specific antagonist. PGI2 and PGE2 were analyzed by ELISA. The results show that DAKD and BK (B1 and B2 agonist, respectively) reduces collagen synthesis in CMF. In presence of GÖ6983 (PKC inhibitor), MAFP (PLA2 inhibitor), indometacin (unspecific COX inhibitor), and rofecoxib (specific COX-2 inhibitor) the decrease in collagen synthesis triggered by DAKD or BK were blocked. However, SC560 (specific COX-1 inhibitor), was unable to prevent the decrease in collagen synthesis induced by DAKD or BK. Both kinins were capable to induce PGI2 but not PGE2 secretion. Furthermore, RO1138452 (IP receptor antagonist), prevents the decrease in collagen synthesis triggers by DAKD. Conclusion: The B1 receptor activation by DAKD decreases collagen synthesis in CMF through PKC-PLA2-COX2-IP signaling pathways.

Dissecting the relevance of the SR-sites posphorylated by Ca2 +calmodulin kinase II (CaMKII) during ischemia reperfusion (I/R) M.N. Di Carlo, C.A. Valverde, M. Said, A. Mattiazzi, M. Salas Centro de Investigaciones Cardiovasculares, CCT-CONICET-La Plata, Facultad de Medicina, UNLP, Argentina Activation of CaMKII exerts a deleterious effect in the irreversible I/ R. CaMKII inhibitors lead to significant decrease of infarct size, necrosis and apoptosis and improve contractility after I/R (Vila-Petroff et al., 2008). SR CaMKII-dependent phosphorylations are involved in the mechanism of injury (Salas et al., 2010). Aim: to elucidate which of the two SR- sites phosphorylated by CaMKII: Thr17 of phospholamban (PLN), or Ser 2814 of RyR2, was responsible for the deleterious effect of I/R. Experiments were performed in two strains of transgenic mice in which either Ser2814 of RyR2 or Thr17 and Ser16 of PLN, were mutated to alanine (Ser2814A and DM, respectively). Isolated Langendorff perfused hearts were submitted to global I/R (45/120 min). Comparing with their controls, DM hearts showed an increase in the infarct area (Ctrl: 20.8 ± 4.4% vs. DM: 44.8 ± 10.7%) together with impaired

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contractile recovery (DM: 44.9 ± 3.5% of control). In contrast, Ser2814A hearts had a significant reduction in the infarct area (Ctrl: 32.1 ± 4.9 % vs. Ser2814A: 16.4 ± 1.6 %, n = 6)) with an improvement in contractility (Ctrl: 2.13 ± 0.49% vs. Ser2814A: 7.66 ± 2.01% of preI), and diminished left ventricular end diastolic pressure during both I and R. Conclusion: 1. CaMKII dependent PLN phosphorylation is beneficial for the post-infarct recovery; 2. CaMKII dependent RyR2 phosphorylation, participates in the deleterious cascade possibly by increasing SR Ca2 + leak during diastole.

Hypotonic swelling promotes nitric oxide release in rat cardiac myoyctes: Impact on swelling-induced negative inotropic effect Luis Gonano, Malena Morell, Martin Vila Petroff Centro de Investigaciones Cardiovasculares CCT-CONICET, Facultad de Cs. Médicas, UNLP, 60 y 120 La Plata, Argentina Cell swelling occurs in multiple pathological situations and in particular, it has been shown to contribute to the deleterious effects of ischemia and reperfusion by promoting contractile dysfunction and predisposing the heart for arrhythmias. We have previously shown that myocyte axial stretch promotes nitric oxide (NO) release. However, whether myocyte swelling promotes NO release and if so, whether it impacts on swelling-induced contractile dysfunction has not been previously assessed. We found that treating rat cardiac myocytes, loaded with the NO sensor DAF-2, with a hypotonic solution, increased cell volume, reduced myocyte contraction amplitude and significantly increased DAF-2 fluorescence. When cells were exposed to the hypotonic solution supplemented with 2.5 mM of the NO synthase inhibitor L-Name, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also inhibited by the PI3K inhibitor Wortmannin. Interestingly, swelling-induced negative inotropic effect was exacerbated in the presence of L-Name or Wortmannin. These results suggest that swelling activates NOS3 (NOS3 being a known target of the PI3K/AKT axis in contrast to NOS1) which promotes NO release. Taken together, these findings suggest a novel mechanism for NO release in cardiac myocytes with putative pathophysiological relevance in the context of ischemia and reperfusion, where it may serve a cardioprotective role by reducing the extent of swelling-induced contractile dysfunction.

Mitochondrial network fragmentation is required for ceramideinduced insulin signaling desensitization in cardiomyocytes C. López-Crisosto, V. Parra, S. Lavandero Center for Molecular Studies of the Cell, Santiago, Chile Obesity is a highly prevalent condition worldwide that represents an important risk factor for the development of diabetes and cardiovascular diseases. Lipid oversupply and lipotoxicity are key factors involved in these complications. It is well known that ceramides, derived from lipid metabolism, contribute to insulin resistance. Cardiomyocytes consume large amounts of ATP, produced through oxidative phosphorylation in the mitochondria, as an energy source for their contractility. Mitochondria form a dynamic network that is constantly remodeled by fission and fusion events. We investigate here the effects of ceramides on insulin signaling and mitochondrial dynamics in cultured rat cardiomyocytes. Treatment of cardiomyocytes with C2-ceramide (40 μM, 3 h) decreased phosphoAkt levels in response to insulin. C2-ceramide also induced mitochondrial dysfunction promoting mitochondrial fission and reducing oxygen consumption. To test whether both effects were linked,

mdivi-1, a chemical inhibitor of the fission protein Drp-1, was added before treatment with C2-ceramide. Mdivi-1 prevented mitochondrial fragmentation caused by C2-ceramide and rescued insulin signaling in cardiomyocytes. In conclusion, the mitochondrial network fragmentation caused by ceramides is required to reduce insulin-dependent Akt phosphorylation in cultured cardiomyocytes. ACT1111, FONDECYT 1120212 and FONDECYT 1090727.

Rho kinase inhibition improves myocardial remodeling in DOCA hypertensive rats D.G. Mondaca, P.A. Araos, C. Yáñez, U. Novoa, I. Mora, M.P. Ocaranza, J.E. Jalil División de Enfermedades Cardiovasculares, Escuela de Medicina, PUC, Santiago, Chile The RhoA/Rho kinase (ROCK) signaling pathway is importantly involved in hypertension as well as in cardiac remodeling. We determined here the effect of fasudil, a direct ROCK inhibitor, on myocardial hypertensive remodeling in DOCA hypertensive rats (50 mg/kg/day for 3 weeks). Results. In fasudil treated rats compared to DOCA hypertensive rats systolic blood pressure and increased cardiac ROCK activity (measuring the levels of phosphorylated to total myosin light chain phosphatase 1 by Western blot) were normalized. Besides, ROCK inhibition with fasudil reduced higher cardiomyocyte area and perimeter (determined by morphometry) by 32 and 17% respectively (p b 0.05) whereas increased myocardial collagen volume fraction was diminished from 16.5% to 4.5% (p b 0.05). At the same time, increased myocardial mRNA levels (determined by RT-PCR) of connective tissue growth factor (CTGF) and p22-phox were both reduced in fasudil treated rats by 41 and 32% respectively (p b 0.05), to levels observed in sham animals. We conclude that in DOCA hypertensive rats, direct ROCK inhibition by fasudil reduces blood pressure and also diminishes cardiomyocyte hypertrophy, myocardial fibrosis and cardiac gene expression of some pro remodeling genes to a significant extent. These effects might occur in hypertension due to other mechanisms as well as in heart failure. Fondecyt 1085208

Participation of TLR4 in post ischemia reperfusion survival in cardiac rat fibroblast P. Ayala, C. Humeres, R. Vivar, M. Catalán, M. Hermoso, G. Díaz-Araya Laboratorio de Farmacoquímica, University of Chile, Faculty of Chemical and Pharmaceutical Sciences, Chile TLR4 receptor is capable of recognizing danger associated molecular patterns present in proteins like HMGB-1, HSP60, HSP70, which are released to interstitial space by necrotic cells triggering an inflammatory response through the ERK 1/2, NF-κB signaling. However, is unknown if TLR4 could participate in the cardiac fibroblast survival post cardiac injury through this same pathways. Methodology: Cardiac fibroblasts were exposed to simulated ischemia for 8 h and 24 h of reperfusion in the presence or absence of TAK 242, intracellular TLR4 inhibitor, the cellular viability was determined by tripan blue counting. Cardiac fibroblast were stimulated with a conditioned medium from cells killed by freezing– thawing in presence or absence of TAK 242 (1 μM), or LPS-RS (5 μg/μL), a TLR4 antagonist. The phosphorylation state of ERK ½ and NF-κB were determined by Western blot. Results: Both TAK-242 and LPS-RS are capable of preventing partially the ERK1/2 and NF-κB activation induced by conditioned

Abstracts

medium. In regard to cellular viability, 8 hour ischemia induces significant cell death, which is enhanced in the presence of TAK 242, this difference is even more marked with 24 h of reperfusion. Conclusion: TLR4 acts as a protective factor in ischemia reperfusion-induced cell death.

TGF-β1 regulates EPAC-1 protein expression in cardiac fibroblasts and myofibroblasts I. Olmedo, M. Catalán, N. Guzmán, M.V. Velarde, G. Díaz-Araya Laboratorio de Farmacoquímica, Departamento de Química Toxicológica y Farmacológica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile EPAC-1 is an effector of cAMP that participates in the repair process post myocardial injury by regulating cardiac fibroblast migration and secretion of extracellular matrix proteins. TGF-β1, is a cytokine that induces cardiac fibrosis; however, it is unknown whether TGF-β1 regulates EPAC-1 expression levels in cardiac fibroblast and myofibroblast, main cells type involved in cardiac fibrosis development. We use primary cultures of neonatal rat cardiac fibroblasts. EPAC-1 expression was evaluated by Western blot. In order to elucidate the involvement of Smad and/or MAPKs pathways activated by TGF-β1, we use specific chemical inhibitors. The results showed that cardiac myofibroblasts have higher EPAC-1 expression levels than cardiac fibroblasts. After 24 h, TGF-β1 reduces EPAC-1 levels in cardiac fibroblasts, while in cardiac myofibroblasts this protein level was increased. In cardiac fibroblast EPAC-1 expression is regulated by JNK and Smad proteins, while in myofibroblast EPAC-1 expression levels is regulated by Smad, JNK, p38, ERK1/2 and Akt signaling pathways. Conclusion: TGF-β1 regulates differentially the EPAC-1 expression levels in cardiac fibroblast and myofibroblast.

Trimetazidine protects cardiomyocytes from mitochondrial fission and dysfunction induced by palmitate J. Kuzmicica, V. Parraa, H. Verdejoa,b, M. Chionga, P. Castrob, S. Lavanderoa a Universidad de Chile, Facultad Cs Químicas y Farmacéuticas, Centro Estudios Moleculares de la Célula, Chile b P. Universidad Católica de Chile, Facultad de Medicina, Santiago, Chile Heart failure (HF) is characterized by alterations in cardiac metabolism and mitochondrial function, and whole body lipid metabolism (increased plasmatic palmitate). Trimetazidine (TMZ) inhibits β oxidation, and our previous findings suggest that it could be used in non-ischaemic HF. In vitro, low concentrations of TMZ induce mitochondrial fusion and increase metabolic parameters, while the treatment with palmitate generates opposite effects. We hypothesized that TMZ has a mitochondrial protective effect in palmitate-treated cardiomyocytes. To test this, isolated cardiomyocytes were treated with BSA-complexed palmitate (25 nM free, 3 h) in the absence or presence of TMZ (1 μM, 24 h). Mitochondrial morphology, by confocal microscopy, oxygen consumption, by Clark electrode, and ATP levels, by a commercial kit, were assessed. Palmitate decreased mean mitochondrial volume (49%, p b 0.05, n = 3), oxygen consumption rate (19%, p b 0.05, n = 3) and ATP levels (16%, p b 0.05, n = 3), while the pretreatment with TMZ protected the cells from these effects. We conclude that TMZ, at low concentrations, has a protective effect in cardiomyocytes against the noxious effect of palmitate. Further

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studies to elucidate the mechanism are needed. ANILLO ACT 1111, FONDECYT 1120212 and 1090727. JK, VP, HV CONICYT scholarships.

The incretin glucagon like peptide-1 (GLP-1) induces mitochondrial fusion and mitochondrial metabolism in vascular smooth muscle cells G. Torresa, P.E. Moralesa, L. Micheab, S. Lavanderoa,b, M. Chionga a Center for Molecular Studies of the Cell, Faculty of Chemical and Pharmaceutical Sciences, Chile b ICBM, Faculty of Medicine, University of Chile, Chile Mitochondrial fusion and fission (mitochondrial dynamics) can control cell metabolism. GLP-1, used in diabetes treatment, promotes glucose metabolism in several cell types. However, its effects on mitochondrial dynamics and metabolism in vascular smooth muscle cells (VSMC) are unknown. Rat aortic VSMC A7r5 were exposed to GLP-1 for different times, loaded with mitotracker orange and the number and area of mitochondria were assessed in multi-slice imaging reconstitutions obtained by confocal microscopy. Mitochondrial potential and ROS were measured by flow cytometry using JC-1 and dihydrochlorofluorescein, respectively. ATP was determined by luciferin/luciferase assay. GLP-1 100 nM for 3 h increased mitochondria area from 0.29 to 0.45 ± 0.04 μm2, suggesting mitochondrial fusion. GLP-1 100 nM for 0.5 h increased mitochondrial potential 1.4 ± 0.2 fold over control. No significant increase in total intracellular ATP content was detected. ROS increase (1.5 ± 0.1 fold over control) was observed only after 6 h GLP-1 exposure. In summary, these results suggest that GLP 1 promotes mitochondrial fusion and metabolism. Supported by FONDECYT 1110180 (MC); ANILLO ACT1111 (MC and SL) and CONICYT thesis support fellowship 24110120 (GT). GT and PM hold a CONICYT fellowship.

Incretin GLP-1 promotes PKA-dependent functional endoplasmic reticulum-mitochondria coupling on vascular smooth muscle cells P.E. Morales, G. Torres, M. Chiong Center for Molecular Studies of the Cell, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Chile Incretin analogs have been successfully used for diabetes treatment. In these patients, vascular smooth muscle cells (VSMC) switch to a highly proliferative, undifferentiated state. Recently, this phenotype was associated with changes on energetic metabolism and increased endoplasmic reticulum (ER)-mitochondria distance. Given that the incretin GLP-1 prevents vascular remodeling, we evaluated whether GLP-1 promoted ER-mitochondria coupling on VSMC line A7r5. GLP-1 100 nM activated PKA and up-regulated Mitofusin-2 levels, an ERmitochondria tethering protein. Immunofluorescence analysis of ERand mitochondria showed that GLP-1 diminished ER-mitochondria distance, as evaluated by an increase on Mander's coefficient. Moreover, treatment with GLP-1 enhanced mitochondrial Ca+ 2 uptake from ER upon histamine stimulation, as visualized by confocal microscopy of cells stained with Rhod-FF. This enhanced coupling of ER-mitochondria seems to be mediated by PKA, given that the PKA inhibitor H-89 prevented the effect of GLP-1. These changes were not correlated with 3H-2-deoxyglucose uptake. These data strongly suggest that GLP-1 promotes functional ER-mitochondria coupling, proposing a novel mechanism for incretin action in VSMC. Fondecyt 1110180 and Anillo ACT1111. PEM and GT hold CONICYT fellowships.

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Abstracts

Mitochondrial fission induced by norepinephrine in cultured cardiomyocytes is counteracts by angiotensin-(1–9) non involving changes in mitochondrial fusion proteins P. Rivera-Mejias, C. Pennanen, C. Sotomayor-Flores, C. Lopez-Crisosto, V. Parra, S. Lavandero Center for Molecular Studies of the Cell, Santiago, Chile Mitochondria form a dynamic network whose morphology and function depend on fusion and fission processes. Previous work from our laboratory has showed that the pathological state of cardiac hypertrophy (CH) triggered by the catecholamine norepinephrine (NE) is accompanied by mitochondrial fission. We have also recently shown that angiotensin-(1–9) is a novel antihypertrophic agent, but it remains unknown whether this peptide may also regulate mitochondrial morphology. Therefore, cardiomyocytes were treated with NE (10 μM) for 24 h with or without angiotensin-(1–9) (100 μM, 0–24 h) and mitochondrial morphology was assessed using Mitotracker. Additionally, the mitochondrial fusion proteins Opa-1 and Mfn2 were assessed using Western blot. Our results depicted that NE induces mitochondrial fission in hypertrophic cardiomyocytes. Moreover, mitochondrial fission generated by NE was prevented by the pre-treatment with angiotensin-(1–9), 6 h. However, angiotensin-(1–9) does not generate changes in either OPA1 or Mfn2, the two proteins involved in mitochondrial fusion regulation. Thus, NE and angiotensin-(1–9) produce opposite effects on mitochondrial dynamics, this being the first report to our knowledge showing that angiotensin (1–9) controls mitochondrial morphology. Anillo ACT 1111, FONDECYT 1120212

Angiotensin (1–9) promotes mitochondrial calcium influx in cultured cardiomyocytes C. Sotomayor-Flores, J. Hechenleitner, P. Rivera-Mejías, C. LópezCrisosto, V. Parra, S. Lavandero Center for Molecular Studies of the Cell, Santiago, Chile Recent findings indicate that the renin–angiotensin–aldosterone system (RAAS) peptide, Angiotensin (1–9), has the ability to antagonize prohypertophic signals in cardiomyocytes. We focus on investigate its effects on calcium signaling, since this ion plays a key role in the contraction and the control of cellular energy metabolism and gene expression in heart. The treatment of cardiomyocytes with 100 μM of Angiotensin (1–9) for 6 h, did not have a direct effect on calcium movements, both cytoplasmic and mitochondrial, but decreased the cytoplasmic calcium response of cardiomyocytes to norephineprine (a pro hypertrophic signal) and increased the mitochondrial calcium transients induced by histamine (which evokes the release of calcium from the endoplasmic reticulum to mitochondria). Finally, Angiotensin (1–9) also recovered the mitochondrial calcium response that is lost in cardiomyocytes pre-treated with norepinephrine, effect that is apparently caused by a decreased coupling between the reticulum and mitochondria. To our knowledge, this is the first study that relates the antihypertrophic effect of Angiotensin (1–9) with the control of the structural and functional coupling between both organelles. Anillo ACT1111 and FONDECYT 1120212 (SL). VP and CLC hold CONICYT fellowships.

The nicotinamide phosphoribosyltransferase inhibitor, FK866, disrupts mitochondrial dynamics and insulin signaling in cardiomyocytes A.P. Oyarzun, V. Parra, R. Troncoso, S. Lavanderos Center for Molecular Studies of the Cell, Faculty of Chemical and Pharmaceutical Sciences University of Chile, Santiago, Chile

FK866, currently in phase II trials, is a promising anti-cancer drug that has been shown to cause apoptosis in several kinds of tumor cells by NAD+ depletion. Since the heart has a highly demanding metabolism, the consequences of a drop in NAD+ levels could result in a severe damaging of this organ. This work is focused on the metabolic changes due the drop in NAD+ levels in primary cultures of neonatal rat cardiomyocytes exposed to FK866. Cell viability was determined by propidium iodide staining. ATP levels by a commercial kit (Cell-Titer Glo, Promega). The effect of FK866 on insulin signaling was studied by Western blot of phosphorylated and total protein levels of insulin receptor (IR), Akt, and mTOR on insulin treated cells. Mitochondrial network integrity was determined by 3D reconstitution of confocal microscopy images of cells loaded with Mitotracker Green®. Our results show that FK866 induces a drop in ATP levels without significant changes in cardiomyocyte viability. However, the drug increased the basal phosphorylation of IR, Akt and mTOR. Insulin was not able to increase the phosphorylation of these proteins. Also, FK866 promotes mitochondrial fusion. Altogether, these results suggest that FK866 impairs cardiomyocytes metabolism. Supported by Anillo ACT1111, FONDECYT 1120212 (SL) and 3110114 (RT)

Cardiac response to β-adrenergic stimulation is partially dependent on mitochondrial Ca2 + uniporter Evaristo Fernández-Sada, Christian Silva-Platas, Cicero Willis, César A. Villegas, Sandra L. Rivero, Cecilia Zazueta, Guillermo Torre-Amione, Gerardo García-Rivas Cátedra de Cardiología, Escuela de Medicina, Tecnológico de Monterrey, Mexico Ca2 + transport through mitochondrial Ca2 + uniporter (mCaU) is the main Ca2 + uptake mechanism in cardiac mitochondria. Despite the importance of mitochondrial Ca2 + to metabolic regulation and mitochondrial function, the consequences of mCaU inhibition on cardiac cell physiology are still poorly understood. We designed the current studies to investigate if even a partial inhibition of mCaU may have a deleterious effect in normal stimulated hearts at baseline and during increased workload due to β-adrenergic stimulation, assuming that an increase in mitochondrial Ca2 + under these conditions is essential for increasing ATP supply. Cardiac contractility and oxygen consumption was measured in ex vivo-perfused hearts from Wistar rats. The addition of Ru360 a selective inhibitor of mCaU induced progressive, sustained negative inotropic effects that were dosedependent with an EC50 of approximately 7 μM. Spermine-treatment a mCaU agonist shown a positive inotropic effect; but was almost blocked by Ru360. In addition, Ru360 decrease β-adrenergic stimulation and did not have effect on PKA signaling. We identified that mCaU inhibition results in a lower β-adrenergic response and in the uncoupling of workload and energetic phosphate metabolites production and overall mimicking energetic conditions in the falling heart.

Cardiovascular Rho kinase (ROCK) activity is related to both leukocyte ROCK activation and apoptosis in rats with high levels of angiotensin converting enzyme (ACE) U. Novoa, V. Orellana, M.O. Ocaranza, J.E. Jalil División de Enfermedades Cardiovasculares, Escuela de Medicina, PUC, Santiago, Chile The intracellular signaling and proremodeling pathway of ROCK is activated in Brown Norway rats (BN; with high ACE and Angiotensin II

Abstracts

levels) compared to Lewis rats. Aim. To assess whether cardiovascular ROCK activity is related to both apoptosis and ROCK activity in circulating leukocytes from BN compared to L rats. Thus, ROCK activity was measured by Western blot levels of phosphorylated/total myosin light chain phosphatase 1, a ROCK target (p-tMYPT1) and apoptosis circulating leukocytes was measured by the TUNEL technique. Results. In BN compared to Lewis rats plasma ACE activity was higher by 78% (p b 0.05) and p-tMYPT1 levels were higher by 122% (p b 0.05) and 160% (p b 0.05) in the left ventricle and aorta, respectively. Simultaneously, in circulating leucocytes from BN compared to Lewis rats p-tMYPT1 levels were higher by 3.7 fold (p b 0.05) whereas apoptosis was higher by 182% (p b 0.05). Conclusions. Cardiovascular ROCK activity is related to both apoptosis and ROCK activity in circulating leukocytes. Besides, increased ACE and angiotensin II levels promote cardiovascular and ROCK activation in circulating leukocytes as well as apoptosis in these cells. Fondecyt 1121060

Rho kinase activity in circulating leukocytes is related to cardiac remodeling and dysfunction in humans J.E. Jalil, M.P. Ocaranza, L. Gabrielli, I. Godoy, P. McNab, U. Novoa, S. Braun, S. Córdova, P. Castro, L. García, M. Chiong, S. Lavandero, I. Mora División de Enfermedades Cardiovasculares, Escuela de Medicina, PUC y Fac. de Cs. Químicas y Farmacéuticas, U de Chile; Santiago, Chile One novel intracellular mechanism that triggers cardiovascular remodeling is the small guanosine triphosphatase Rho and its target Rho kinase (ROCK). We hypothesized here that in humans, ROCK activation in circulating leukocytes (assessed by the levels of phosphorylated to total myosin light chain phosphatase 1; MYPT1P/T), is related to pathological cardiac remodeling in a direct way. Control subjects (n = 51); untreated hypertensive patients with LVH (n = 41) and patients with stable congestive heart failure under full treatment (HF, n = 17) were compared. LV ejection fraction (EF) and LV mass index were determined by 2D echocardiography. Results: as means ± SEM; # = p b 0.01 (ANOVA).

EF (%) LVMI g/m2 MYPT1-P/T

Controls

HT + LVH

HF

62.9 ± 0.3 86.2 ± 2 2.1 ± 0.4

61.2 ± 0.5 125 ± 4 19 ± 4

27 ± 2 # 160 ± 12 # 320 ± 169 #

Thus, in humans, as both cardiac remodeling and LV dysfunction evolve, ROCK in circulating leukocytes is progressively activated. ROCK activity in circulating leukocytes might be used as a possible biomarker of cardiac remodeling and failure. Fondecyt 1085208 and 1121060

Angiotensin-(1–9) decreases blood pressure and protects vascular endothelium in hypertensives rats with low renin N. Escudero, J. Moya, V. Barrientos, C. Morales, U. Novoa, M. Chiong, S. Lavandero, L. Michea, J. Jalil, M.P. Ocaranza Previously, we have demonstrated that angiotensin (Ang)-(1–9) decreases blood pressure and cardiovascular remodeling on hypertension. There is no evidence about the effect of Ang1-9 in hypertensive rats with low renin. Objective: To determine the effect of the chronic administration of Ang1-9 on blood pressure and vascular endothelium in hypertensive rats with low renin. Methods: The deoxycorticosterone acetate (DOCA)-salt hypertensive model was used in Sprague–Dawley male rats aged 5–6 weeks (150 ± 10 g). DOCA (60 mg/kg, im) twice a week was administered starting

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immediately after recovery of surgery. The animals received 1% NaCl and 0.4%KCl in the drinking water. As controls, uninephrectomized rats were used (Sham group, n = 12). At the second week, the hypertensive DOCA-salt rats were randomized to receive vehicle (n = 10), Ang-(1–9) (602 ng/kg/min, n = 12) and Ang-(1–9) and Mas blocker receptor A779 (100 ng kg− 1 min− 1,n = 7) for 2 weeks. Systolic (S) and diastolic (D) blood pressure (BP), body mass (BM), heart weight (HW), heart mass relative to the length of the tibia (HMR), plasma renin activity (PRA), lumen area of the aortic wall (LA), vascular reactivity Assays (VRA,% dilation at 10− 9 M AcCh), and eNOS mRNA levels (fold) in the aortic wall were determined. Results: The hypertension significantly increased SBP (66%), DBP (60%), HW (30%), HWR (36%) and decreased PRA (96%) and VRA (87%) compared with Sham rats. Ang1-9 significantly decreased SBP (22%), DBP (25%) and increased LA(15%), VRA(12 fold), eNOS (55%). These effects were not mediated by Mas receptor Conclusion: Ang 1– 9 decreased blood pressure and protected vascular endothelium in hypertensive rats with low renin. These effects were not mediated by Ang-(1–7). Fondecyt 1100874, Fondef 11I1122

The antihypertensive effect of angiotensin-(1–9) is mediated by kidney sodium handling J. Moyaa, V. Barrientosb, C. Moralesa, U. Novoaa, N. Escuderoa, M. Chiongc, S. Lavanderoc, L. Micheab, J. Jalila, M.P. Ocaranzaa a Enf. Cardiovasc, P. U. Católica de Chile, Chile b Lab. Fisiol., Fac. Medicina, Chile c Cs Químicas, U Chile, Chile We observed that angiotensin (Ang)-[1–9] exerts anti-hypertensive and vascular protective effects in hypertensive rats. However, it is unknown whether the anti-hypertensive effect is associated with kidney sodium handling. Objective: To determine whether the antihypertensive effect of Ang (1–9) is associated with a diureticnatriuretic mechanism. Male Sprague Dawley rats to 200 ± 10 g were randomized to receive Ang II (400 ng/kg min) by osmotic pump. As controls we used sham operated rats (n = 18). Two weeks after surgery sham and hypertensive rats were randomized to receive vehicle, (n = 18), Ang-(1–9) (602 ng/kg/min, n = 17), Ang-(1–9) and Mas blocker receptor A779 (100 ng kg− 1 min− 1,n = 7) for 2 weeks. Systolic blood pressure (SBP), relative cardiac mass (RCM), cardiomyocytes area (CA) and perimeter (CP) and cardiac fibrosis in the left ventricle (by collagen volumetric fraction, CVF) were determined. To evaluate the diuresis and natriuresis, normotensive rats were randomized to receive either vehicle (n = 8) or Ang-(1–9) (600 ng kg− 1 min− 1, n = 8) for 6 days. Daily urine samples were obtained for 5 days, starting 24 h after minipump implantation. Urinary sodium concentration was determined. Hypertensive rats compared to sham rats had a significant increase (p b 0.05) of SBP (33%), RCM (17%), CA (64%), CP (26%), CVF (46%). The chronic Ang(1–9) administration decreased SBP (20%), RCM (13 %), CA (35%), CP (20%) and CVF (20%). These effects were not mediated by Mas receptor. As compared to vehicle-infused rats, Ang-(1–9) showed a significant increase in diuresis at days 2, 3, and 6 and significant increase in natriuresis at days 2 and 3 of infusion with Ang-(1–9). Conclusion: Ang-(1–9) reduces hypertension and myocardial remodeling in hypertensive rats. In healthy rats showed that the acute infusion with Ang-(1–9)-induced diuresis and natriuresis. This is the first report showing the effect of Ang-(1–9) in the regulation of renal sodium. New therapies aimed at increasing Ang-(1–9) plasma levels may be useful in the treatment of hypertension and reducing endorgan damage. Fondecyt 1100874, Fondef 11I1122

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Abstracts

The vasodilator effect of angiotensin-(1–9) is mediated by the AT2 receptor and by increasing nitric oxide J. Moyaa, V. Barrientosb, L. Garcíac, U. Novoaa, M. Chiongc, S. Lavanderoc, L. Micheab, J. Jalila, M.P. Ocaranzaa a Enf. Cardiovasc, P. U. Católica de Chile, Chile b Lab. Fisiol., Fac. Medicina, Chile c Cs Químicas, U Chile, Chile Angiotensin (Ang)-[1–9] prevents hypertension (HT) and exerts cardioprotective effects in the heart probably by antagonizing Ang II effects. We hypothesized here that Ang-(1–9) reduces HT and vascular remodeling through the AT2 receptor and through eNOS. Objective. To determine the antihypertensive and vascular effects of Ang-(1–9) in hypertensive rats and the signaling pathways involved in Ang-(1–9) effects. Male rats were made hypertensive by Ang II infusion or Goldblatt procedure (n = 46). As controls we used sham operated rats (n = 26). Two or four weeks after infusion or surgery, respectively, the hypertensive rats were randomized to receive vehicle, n = 22, Ang-(1–9) (602 ng/kg/min, n = 24), Ang-(1–9) and Mas blocker receptor A779 (100 ng kg− 1 min− 1, n = 15) and Ang-(1–9) and AT2 receptor blocker PD123319 (36 ng kg− 1 min− 1, n = 14). Four and six weeks after surgery, systolic blood pressure (SBP, mmHg), aortic media thickness (AMT, mm), TGFβ1 and collagen I aortic (CA) levels (fold vs S), vascular reactivity Assays (VRA, % dilation at 10- 9 M AcCh), eNOS mRNA levels (fold) and plasma nitrate concentrations (PNC, μM) were determined. Hypertensive rats compared to sham rats had a significant increase (p b 0.05) of SBP (50%), AMT (35%), TGFb1 (2 fold), CA (4 fold), and decrease of VRA (5%) and PNC (40%). The chronic Ang-(1–9) administration decreased SBP (18%), AMT (10%), TGFb1 (1.9 fold), CA (2 fold) and increased VRA (20%), and PNC (1.6 fold). These effects were reverted by PD123319 but not by A779. Conclusion. The current study provides strong evidence of antihypertensive and antiremodeling effects of Ang-(1–9) are mediated by the AT2R and eNOS. Fondecyt 1100874, Fondef D11I1122.

L-type calcium current modulation by angiotensin II T. Hermosillaa, C. Morenoa, E. Salamancaa, S. Linsambartha, D. Sarmientob, F. Simonb, D. Varelaa a CEMC & ICBM, Facultad de Medicina, Universidad de Chile, 838-0453, Santiago, Chile b Facultad de Ciencias Biológicas y Facultad de Medicina, Universidad Andres Bello, 837-0134, Santiago, Chile The L-type calcium channel is formed by the association of the pore subunit CaV1.2 and the accessory subunits CaVβ and CaVα2δ1. In heterologous systems, the magnitude of L-type Ca2 + current inhibition after angiotensin II (AngII) exposure is dependent on the CaVβ subunits expressed, with CaVβ1b containing channels being strongly regulated and CaVβ2a containing channels being almost insensitive. In the normal heart the most abundant CaVβ subunit expressed is the CaVβ2b splice variant at variance with the hypertrophic heart which over-expresses the CaVβ2a splice variant. Therefore, we explored a possible differential effect of AngII over L-type calcium currents depending on the heart's physiological state. Using rat neonatal cardiomyocytes cultures from different days of culture as an in vitro model for hypertrophy, we measured the response of L-type calcium currents to AngII exposure. The role of CaVβ2 splice variants on AngII effect was studied in cardiomyocytes transduced with both variants. Interestingly, CaVβ2a-transduced cardiomyocytes showed almost no sensitivity to AngII while CaVβ2b-transduced cardiomyocytes were strongly inhibited. Our results suggest that AngII-dependent calcium homeostasis in a hypertrophic heart is

different compared to a healthy heart and could reflect a novel mechanism that may contribute to the understanding of the deleterious effects of AngII.

Reversibility of redox changes in cardiac ryanodine receptor J.P. Finkelstein, G. Sánchez, R. Bull, P. Donoso Physiology and Biophysics Program and CEMC, Facultad de Medicina, Universidad de Chile, Chile Ryanodine receptors (RyR) incorporated in planar bilayers exhibit low (L), moderate (M) or high (H) activity in response to cytoplasmic calcium depending on the redox state of some highly reactive SH residues of the protein. Little is known about the incidence of RyR redox changes or its reversibility in vivo. In this work we show that rat hearts perfused with Krebs solution contain less active RyR than blood perfused hearts from rabbit (37% L, 58% M and 5% H channels in Krebs vs 0% L, 50% M and 50% H channels in rabbit hearts), suggesting a more reduced state of the channel protein in Krebs perfused rat hearts. In Krebs perfused hearts, 5 min of global ischemia followed by 1 min of reperfusion, changed drastically the profile of channel activity, decreasing L channels to 10% and increasing H channels to 37%. Fifteen minutes of reperfusion after ischemia was sufficient to fully revert this change: the profile of channel activity was indistinguishable from controls. Incubation of RyR enriched vesicles isolated from controls with NADPH and GSH, to generate ROS through NADPH oxidase and to Sglutathionylate oxidized SH residues, increased the frequency of appearance of H channels. Addition of glutaredoxin reverted this effect. We conclude that redox changes occur transiently ex vivo and are presumably reverted by glutaredoxin. Fondecyt 1110257 and Fondap 1501006

β-adrenergic stimulation causes rapid NOS1-dependent Snitrosylation (S-NO) of cardiac proteins A.Z. Vielmaa, M.P. Borica, D.R. Gonzálezb a Dept. Fisiología, FCB, P. Universidad Católica de Chile, Santiago, Chile b Dept. Ciencias Básicas Biomédicas, FCS, Universidad de Talca, Talca, Chile Neuronal nitric oxide synthase (NOS1) increases heart inotropism, but the mechanisms remain controversial. We hypothesized that βadrenergic stimulation induces NOS-1-dependent S-NO of Ca2 + regulatory proteins. In the isolated rat heart, isoproterenol (ISO, 10 nM, 3-min) increased S-NO of the ryanodine receptor (RyR2) (+146 ± 77%) and total proteins (+46 ± 14%), without affecting SNO of SERCA2 and the L-Type Ca2 + channel (LTCC). Selective NOS1 inhibition with S-methyl-L-thiocitrulline (SMTC) decreased basal SNO of total proteins, RyR2, SERCA2 and LTCC (− 60-75%), besides reducing contractility and relaxation. SMTC also prevented ISOinduced S-NO of RyR2 (−85 ± 7%), without affecting signaling via phospholamban phosphorylation. NOS1 blockers SMTC and Nωpropyl-l-arginine (LNPA), decreased basal contractility (−25-30%) and cardiac protein S-NO (−25-60%), and reduced (−25-40 %) ISOinduced inotropic response and protein S-NO; and all these effects were mimicked by Tempol, a superoxide scavenger, inhibitor of S-NO. In contrast, selective NOS3 inhibitor L-NIO did not modify inotropism or protein S-NO. We conclude that basal NOS1 dependent S-NO of the calcium-cycling machinery is required for optimal cardiac regulation and β-adrenergic stimulation induces rapid NOS1dependent S-NO, accounting for about one third of the cardiac reserve. Grants Fondecyt 1090757 & 1120595.

Abstracts

NOX2 inhibition restores contractility in dytrophic cardiomiopathy D.R. González, A.V. Treuer, R.A. Dulce, J.M. Hare Universidad de Talca, Chile and University of Miami, USA Dystrophic cardiomyopathy is the cardiac manifestation of Duchenne dystrophy. Oxidative stress is characteristic of cardiomyopathies and intracellular calcium ([Ca2 +]i) handling is abnormal in the heart of the mdx mouse, a model of Duchenne muscular dystrophy. We tested the hypothesis that oxidative stress may be responsible of disturbances in Ca2 + handling and contractility in this model. We found increased expression (fivefold) of the NADPH oxidase NOX2 in the mdx hearts compared to wild type, along with increased superoxide production, (p b 0.05 vs. wild type). Treatment with apocyinin, a NOX2 inhibitor, decreased superoxide in mdx (p b 0.05 vs. untreated). Next, we studied the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility, was decreased in mdx myocytes compared to wild type (p b 0.05). Pre-treatment with apocynin restored this response to normal levels. In addition, the amplitude of evoked [Ca2 +]i transients (measured as fura-2 fluorescence) that was diminished in mdx myocytes (p b 0.05), was also restored upon NOX2 inhibition. These results indicate that in mdx hearts, NOX2 inhibition reduces oxidative stress, improving the SR [Ca2 +] handling and contractility. Targeting of NOX2 in may be therapeutically helpful to increase cardiac performance Duchenne and other dystrophic cardiomyopathies.

Impact of post-translational modifications of cardiac ryanodine receptor (RyR2) on reperfusion arrhythmias M. Said, R. Becerra, A. Herrero, M. Tellechea, C. MundiñaWeilenmann, A. Mattiazzi, L. Vittone Centro de Investigaciones Cardiovasculares, CCT-LA Plata, CONICETUNLP, Argentina Reperfusion (R) after ischemia is particularly prone to cardiac arrhythmias. This work investigated the role of redox modifications and CaMKII-dependent phosphorylation (Ser2815 residue) of RyR2 upon arrhythmias appearance. Langendorff perfused rat/mouse hearts were submitted to global ischemia/reperfusion. Epicardial monophasic action potentials and left ventricular developed pressure were recorded. Premature beats (PB) were counted during R in the absence or the presence of inhibitors: 10 μM APO (NADPHox), 10 μM L-Name (NOS) and 2.5 μM KN-93 (CaMKII). At the onset of R, S-glutathionylation (SGLU), S-nitrosylation (S-SNO) and Ser2815 phosphorylation of RyR2 increased. APO significantly decreased S-GLU of RyR2 and increased PB from 42 ± 4 to 59 ± 3. L-Name did not modify PB and KN-93 significantly decreased both Ser2815 phosphorylation and PB to 13 ± 3. The results suggest that CaMKII-phosphorylation of RyR2 at the onset of R favours arrhythmias, whereas S-GLU-RyR2 may partially prevent them. It would be interesting to explore if the spontaneous SR Ca2 + leak induced by phosphorylation, main mechanism responsible for arrhythmias, is limited by S-GLU-RyR2.

β-adrenergic effect on Ca2 + affinity and cross-bridge cycling rate in a cardiac contractile model J.A. Negronia, J.L. Puglisib, E.C. Lascanoa, A.V. Gomesb, D.M. Bersb a Universidad Favaloro, Buenos Aires, Argentina b University of California Davis, CA, USA β-adrenergic stimulation (β-AS) phosphorylates myocyte troponin I (TnI) and myosin-binding protein C (MyBP-C). However, their relative

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contribution to decreased-Ca2 + sensitivity and increased cross-bridge (XB) cycling rate in β-AS force generation is unclear. A Ca2 +-driven 5 state force model integrated into a well-established myocyte model was used to analyze force response to separate β-AS stimulation on myofilament Ca2 + sensitivity (XBCa) and XB cycling rate (XBcy). The model reproduced experimental β-AS effects on the force-Ca2 +, release–restretch, length–step, stiffness–frequency and force–velocity relationships. β-AS increased peak Ca2 + by 61% and isometric force by 46% and reduced relaxation time by 36%. Then, individual β-AS effects were switched-off to analyze their impact. Removal of β-AS effect from XBCa decreased peak Ca2 + and increased contractile force by 115% with respect to control, while β-AS elimination from XBcy nearly eliminated the effect from force, preserving β-AS increase in Ca2 +. Satisfactory reproduction of experimental reports suggests separate XBCa and XBcy effects on force response to β-AS. Moreover, increase in XBcy is essential for β-AS -induced inotropy, because the reduced myofilament sensitivity and increased Ca2 + transient are nearly offsetting with respect to contractile force.

Biomarkers of oxidative stress in a model of isolated heart in rats: Effect of supplementation with omega-3 fatty acids R. Castillo, R. Rodrigo, G. Sanchez Pathophysiology Program, Faculty of Medicine, University of Chile, Chile Increased levels of ROS (reactive oxygen species) have been demonstrated during ischemia and post-ischemic reperfusion in acute myocardial infarction in humans. The aim of this work was to assess the oxidative stress related parameters in a rat model of AMI and the effects of omega 3 supplementation in this biomarkers and the infarct size. Male SD rats (n = 15) that weight approximately 250–350 g will be supplemented with n − 3 PUFA in two groups: G1 (n = 5); 0.6 g/kg/day and G2 (n = 5), 1.2 g/kg(day for 8 weeks. Basal group included 5 rats exposed to standard diet. Experiments will be performed in isolated hearts perfused in conditions of ischemia 30 min and reperfusion 120 min according to the Langendorff technique. Hemodynamic registration was tabulated in each group. Infarct size, TBARS content, GSH/GSSG ratio, antioxidant enzymes activities (catalase-CAT, glutathione peroxidase-GSH-Px and superoxide dismutase-SOD) and nuclear factor kappaB activation in these samples were determined. The infarct size was reduced in 30.7 and 35.3% in G1 and G2, respectively (p b 0.05). The GSH/GSSH was 5.3 and 9.7 fold higher in both groups, and TBARS showed a marked reduction in G1 at reperfusion 120 min (p b 0.05). SOD activity was 33.5 and 49.8% higher in G1 and G2, respectively at ischemia 30 min (p b 0.05). No significant differences were found in the other enzymes. With respect to NF-kappaB activation in G1 and G2, this showed 22.5 and 30.5% lower DNA binding than that of basal conditions at reperfusion 120 min (p b 0.01). These results would postulate that the cardioprotective effects of omega-3 may be mediated through attenuation of oxidative and inflammatory damage in myocardial tissue. FONDECYT 11110426.

The antiarrhythmic effect of fish oils in a mouse model of catecholaminergic polymorphic ventricular tachycardia (CPVT) V.C. De Giusti, L. Venetucci, J.E. Avelino Cruz, F. Lodola, A. Curcio, R. Bongianino, M. Denegri, N. Monteforte, R. Bloise, C. Napolitano, S.G. Priori Fondazione Salvatore Maugeri I.R.C.C.S. Pavia, Italy

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Abstracts

The autosomal dominant form of CPVT, caused by mutations in the ryanodine receptor (RyR2), is characterized by the onset of spontaneous Ca release (SCR) from the sarcoplasmic reticulum that leads to delayed after-depolarizations (DADs) and arrhythmias during adrenergic stimulation. We investigated the antiarrhythmic effect of Fish Oils (DHA and EPA) in heterozygous RyR2/RyR2R4496C mice, both in vitro (with patch-clamp) and in vivo (with ECG recordings). In vitro preincubation with DHA 10 μM or EPA 20 μM prevented the Isoinduced SCR delayed and DADs (Iso: 71%; DHA: 10.5%; EPA: 6%) and triggered activity (TA) (Iso: 68%; DHA: 5.5%; EPA: 0%). L-type calcium current (ICaL) was reduced (control: − 5.4 ± 0.6, n = 25; DHA: − 3.6 ± 0.5, n = 21; EPA: −3.7 ± 0.3, n = 15) and the Iso-induced ICaL stimulation was prevented by DHA or EPA (Iso: −9.4 ± 1; n = 14, DHA: v2.6 ± 0.5; n = 15, EPA: − 4.3 ± 0.4; n = 15). The SR luminal threshold for calcium leak (53.5 ± 3.2 μM; n = 5) was enhanced by DHA (70 ± 4.2 μM; n = 4) and EPA (83 ± 6.3 μM; n = 11). Consistently, in vivo bidirectional ventricular tachyarrhythmias were prevented by Fish Oils administration. These results suggest that the antiarrhythmic effect of Fish Oils is likely due to the modulation inhibition of both ICaL and RyR2. Moreover, the in vivo data, allow us to propose Fish Oils as an alternative or additional treatment for the CPVT patients.

Proteasome inhibition protects the heart from ischemia–reperfusion injury J. Pezoa, L. Montecinos, G. Sánchez, P. Donoso ICBM, Facultad de Medicina, Universidad de Chile, Chile Contractile dysfunction following cardiac ischemia reperfusion (IR) is related to the extent of protein degradation during the ischemic period and subsequent reperfusion. Alterations in the activity of the proteasome, a major cellular proteolytic pathway, may produce the uncontrolled degradation of functional proteins and further enhance the damage induced by IR. Inhibition of the proteasome during IR has produced inconsistent results; there are reports of both, beneficial and adverse effects. Discrepancies may be due in part to the very high concentrations of inhibitors used, which may elicit other non specific responses. The aim of this work was to study the effect of a low concentration of the proteasome inhibitor MG132 (0.5 μM) on myocardial contractile parameters after IR using a Langendorff perfused rat heart model. We found that MG132 administered before 30 min of global ischemia followed by 60 min of reperfusion significantly improved the recovery of the left ventricular developed pressure (IR: 14 ± 6 %, n = 6; IR + MG132: 51 ± 28 %, n = 5), + dP/dt (IR: 8 ± 4 %, n = 6; IR + MG132: 48 ± 28 %, n = 5) and –dP/dt (IR: 13 ± 6 %, n = 6; IR + MG132: 58 ± 32 %, n = 5). MG132 also decreased diastolic pressure after IR. We conclude that non toxic inhibition of the proteasome reduces IR injury. Fondecyt 1110257, Fondap 1501006

Boldine reduces cardiac I/R injury R. Hernandez, A. Vielma, M.P. Boric, J.C. Saez, V. Velarde Faculty of Cs. Biology, Department of Physiology, Catholic University of Chile, Chile Cardiac ischemia/reperfusion (IR) injury is the leading cause of death in developed countries. In IR proinflammatory factors and reactive oxygen species are increased, while contractility decreases.

Deregulation of gap junction cell–cell coupling and increase in connexin hemichannels (Cx-HC) activity may contribute to injury progression. Boldine is a potent antioxidant and blocker of Cx-HC, thus, we postulate that it may help prevent heart damage in IR. Isolated hearts of healthy SD rat, were perfused with control buffer, 10 μM boldine, or 10 μM ascorbic acid (AA), used as an antioxidant control, and submitted to 50-min ischemia plus 40-min reperfusion. We measured heart rate, cardiac contractility, work index (WI), and cell permeability by the uptake of ethidium (Et-U). IR caused a decrease in contractility (− 42%) and WI (−58%) and increased Et-U (6.7-fold), relative to control, non-ischemic hearts. In contrast, IR did not produce significant reduction in WI or contractility in hearts treated with AA or boldine. However AA-treated hearts showed a declining tendency within the observation period. In addition, Et-U was reduced in boldine-treated (3.4-fold), but not in AA-treated heart (8.9-fold). We conclude that boldine prevents the decline in cardiac function in IR, not only by its antioxidant properties but also by its Cx-HC blocking effect. Grants Fondecyt Anillos ACT-71 and Fondef D0I-1086.

Myocardial superoxide anion production induced by angiotensin II: Mineralocorticoid-receptor (MR)-dependent and independent fraction C.I. Caldiz, G.E. Chiappe de Cingolani, H.E. Cingolani The RAA system induces superoxide anion (O 2 −) production by a NADPHoxidase (NOX) dependent mechanism. Here we compare the effects of Ang II 1 and 100 nM on the production of O 2− and the intracellular involved mechanisms. Strips from rat cardiac muscles were used to measure O 2− production by luminescence with lucigenin and the results were expressed as % of control. Ang II induced an increase of O 2− production in a dose dependent manner. At 1 nmol/L the increase was (50.6 ± 2.5%) effect that was blunted by Bosentan (BT − 3 ± 3.9%) and BQ123 (11 ± 4.1%). The increase was abolished by inhibiting NOX with apocynin (Apo 9.09 ± 10.9%), MR with Spironolactone (Sp 8.5 ± 8%), the EGFR with AG 1478 (AG-1.7 ± 8.8%) and the matrix metalloproteinases (MMP) with MMP1 (−3.1 ± 8.9%). The increased was also blunted by inhibiting KATP channels with (5HD (1.4 ± 10.9%) or glibenclamide (Gli −7.7 ± 6.5), the mitochondrial respiratory chain with rotenone (Rot 10.4 ± 9.3%) or the MTP with cyclosporyn (CsA 5.1 ± 3.3 %) or bongkrekic acid (BK 2.5 ± 5.3%).The O 2− production induced by 100 nmol/L of Ang II was also abolished by Apo (9.8 ± 9.4%) and by the mitochondrial inhibitors 5HD (15.8 ± 8.6%), Gli(−6 ± 6.6%), Rot (4.1 ± 3.71%), CsA (11.4 ± 8.4%) and BK (3.6 ± 4.5%) but only decreased in part by BT (32.1 ± 8.8%) and BQ(37.1 ± 7.9%), Sp (31.4 ± 10.8%), AG1478 (52.0 ± 8.8%) and MMP1 (43.4 ± 11.5%). Both doses of AngII increased the O 2 − production from mitochondrial source and by a NOX-dependent mechanism. 1 nmol/L AngII needs an active ET1 and MR, activation of MMP and EGFR transactivation. At higher doses of AngII (100 nmol/L) there is a fraction of O 2 − production from mitochondrial origin that involves other signalling pathways independent of MR or EGFR activation.

Simulation of postacidotic arrhythmias in a human myocyte model. Role of CAMKII E. Lascanoa, M. Saidb, L. Vittoneb, A. Mattiazzib, C. MundiñaWeilenmannb, J. Negronia

Abstracts a

Universidad Favaloro, Buenos Aires, Argentina Centro de Investigaciones Cardiovasculares, CONICET, La Plata, Argentina

b

Postacidotic arrhythmias are associated to increased sarcoplasmic reticulum (SR) Ca2 + load and Ca2 +/calmodulin-dependent protein kinase II (CaMKII) activation, but the underlying mechanisms are still unclear. To understand this process, acidosis effects were incorporated into a myocyte model of contractility, including acidotic inhibition of L-type Ca2 + channel (ICaL), Na+-Ca2 + exchanger, Ca2 + release through SR ryanodine receptor (RyR2) (Irel), Ca2 + reuptake by the SR Ca2 + ATPase2a (Iup), Na+–K+ pump and K+ efflux and increased activity of the Na+–H+ exchanger (INHE). CaMKII effects on Irel, Iup, ICaL, INHE and late INa were introduced to partially compensate acidosis as experimentally reported. Assuming that diastolic Ca2 + leak through RyR2 was modulated by the resting state of this channel, postacidotic delayed afterdepolarizations (DADs) were triggered upon returning to normal pH after 6 min acidosis at 30% CO2-induced pH = 6.7. The model confirmed that DADs depend on Ca2 + leak from Ca2 + overloaded SR and CaMKII activation, as their inhibition abrogated DADs. The model further revealed that in the transition to normal pH, DADs are determined by SR Ca2 + load and thereafter maintained by SR reloading with Ca2 + influx through the reverse NCX mode during the time period in which [Na+]i is elevated.

The balance between sarcoplasmic reticulum (SR) Ca2 + uptake and release is critical to determine the effects of Epac activation in the myocardium N. Lezcano, I. Lucotti, M. Said, L. Vittone, C. Mundiña-Weilenmann Centro de Investigaciones Cardiovasculares, CONICET-La Plata, UNLP, Argentina In cardiac muscle, the role of the exchange protein directly activated by cAMP (Epac) in the regulation of Ca2 + homeostasis and contractility, and the signalling pathways involved in these effects are controversial. To elucidate the reason for the discrepancies, the effects of a selective activator of Epac, 8-CPT, were studied in isolated adult rat myocytes loaded with Fura-2 AM at different extracellular Ca2 + ([Ca]o). 10 μM 8-CPT significantly increased fractional sarcomere shortening (134.6 ± 15.5% of control, n = 13), Ca2 + transient amplitude (116.6 ± 6.4% of control, n = 13) and SR Ca2 + content from 0.51 ± 0.03 to 0.62 ± 0.04 F340/380 (n = 6) at 0.5 mM [Ca]o. At 1 mM [Ca]o no changes were observed and at 1.8 mM [Ca]o, stimulation of the Epac reduced all three parameters (p b 0.05). At all [Ca]o, activation of Epac increased CaMKII-dependent phosphorylation of phospholamban, which by enhancing SR Ca2 + uptake could be responsible for the increased SR Ca2 + content. At higher [Ca]o, 8-CPT increased CaMKII-dependent phosphorylation of the ryanodine receptor in association with an increased SR Ca2 + leak. The results suggest that Epac activation, recruits a CaMKII pathway, independently of PKA, which exerts both positive and negative inotropic effects depending on the balance between SR Ca2 + uptake and release.

Cardiac calcium handling in Fabry disease C.A. Valverde, L. Gonano, J. Mucci, G. Rinaldi, P. Rozenfeld, M. VilaPetroff, A.M. Mattiazzi Centro de Investigaciones Cardiovasculares, La Plata, BsAs, Argentina Fabry (Fb) disease is a genetic X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A that affects, inter alia, to

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cardiac tissue. Clinical evidence indicates that cardiac arrhythmias are common in older patients with Fabry disease, having a significant impact on patient survival. We assessed whether there is a potential arrhythmogenic substrate at the cellular level responsible for triggering the cardiac arrhythmias. A murine model of Fb disease (KO mice) and WT mice were used. Homogenates from freshly isolated hearts from both strains were used for Western blotting for calcium handling proteins. Isolated myocytes were employed for evaluating contractility, intracellular Ca transients and SR calcium content. Ca sparks and waves were assessed by confocal microscopy. Hearts from Fb mice exhibited a similar protein expression level of CaMKII, NCX, PCaMKII, SERCA2a and RyR2. However, there was a significantly increased phosphorylation of RyR2 in the PKA and CaMKII sites in Fb respect to WT (193.3 ± 22.8% vs. 100.0 ± 12.4%, 232.5 ± 55.8% vs. 100.0 ± 14.8%, respectively). This increase was not accompanied by a significant alteration in SR calcium content but was associated with a significant increase in calcium sparks and waves (in absence and presence of Iso), with respect to WT mice. The increase in RyR2 phosphorylation in Fb mice hearts could be the basis for the higher number of sparks and waves observed in this disease. This provides an arrhythmogenic substrate either trigger or maintain arrhythmias in Fabry disease.

GSK3-β/NFAT signaling is involved in testosterone-induced cardiomyocyte hypertrophy J. Durán, N. Venegas, C. Oyarce, M. Pavez, R. Maass, M. Estrada ICBM, Facultad de Medicina, Universidad de Chile, Chile In cardiomyocytes testosterone induced hypertrophy through androgen receptor-dependent and -independent signaling pathways. Glycogen Synthase Kinase 3-β (GSK3-β) is a negative regulator for cardiac hypertrophy. GSK3-β controls gene expression mainly via activation of the nuclear factor of activated T cells (NFAT). However, the role of the GSK3-β/NFAT axis on testosterone-induced hypertrophy is unknown. Here we have studied whether testosterone effects on GSK3-β and NFAT participate in the cardiomyocyte hypertrophy. Testosterone (100 nM) increased the NFAT activity, which was blocked by NFAT inhibitors cyclosporine A and FK506. Moreover, testosterone inhibited GSK3-β (phosphorylation increase at Ser9). Inhibition of PI3K/Akt by LY-292002 and Akt-inhibitor-VIII blocked testosterone-induced GSK3-β phosphorylation, whereas ERK1/2 inhibition had no effect. Long-term stimulation with testosterone increased cell size and expression of skeletal α-actin and β-myosin heavy chain. Inhibition of androgen receptor did not modify increase in GSK3-β phosphorylation but inhibited hypertrophy. Transfection of cardiomyocytes with a plasmid expressing a constitutively active form of GSK3-β (GSK3βS9A) decreased NFAT activity and hypertrophy induced by testosterone. Moreover, pharmacological inhibition of GSK3-β increased cardiomyocyte hypertrophy, which was not further increased by testosterone stimulation. These results suggest that testosterone inhibits GSK3-β and activates NFAT, which could contribute to cardiomyocyte hypertrophy. FONDECYT 1120259

Left ventricular sexual dimorphism: When does it start? D. Broda, C. Massarutti, A. Díaz, O. Pinilla, I. Ennis, E. Escudero Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, UNLP, CONICET, La Plata, Argentina The present work aims to analyse left ventricular structural and functional characteristics in a group of individuals of both sexes in order to identify when the differences appear as well as the possible

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Abstracts

mechanisms involved. 302 subjects, 160 women (w) between 6 and 24 years old were studied obtaining anthropometric parameters, blood pressure and echocardiogram. Males had higher systolic blood pressure (SBP), stroke volume (SV) and left ventricular mass (LVM) than women from the age of 13 as shown in the table. Differences in SBP, SV and BSA accounted for 73% of the dimorphism in the LVM, remaining a 27% of an undetermined origin when adjusting for these previous variables. Interval

SBP (mmHg) Women

6 to 107.53 ± 2.01 12 years 13 to 112.47 ± 0.82 24 years Interval

BSA (m2) Women

6 to 1.03 ± 0.06 12 years 13 to 1.60 ± 0.01 24 years

SBP (mmHg) Men

pb

108.64 ± 1,88 ns

LVM (g) Women 62.05 ± 5,73

LVM (g) Men

pb

64.36 ± 3.81 ns

123.79 ± 1.01 0.01 102,51 ± 1,67 141.50 ± 2.76 0.01 BSA (m2) Men

pb

SV (ml) Women

SV (ml) Men

1.12 ± 0.04

ns

43.94 ± 2.78

49.57 ± 2.39 ns

1.84 ± 0.02

0.01 59.11 ± 1.00

pb

73.28 ± 1.37 0.01

Left ventricular sexual dimorphism was evident from 13 years old, with higher LVM in males. Even though the differences in SBP, SV and BSA partially justify it, there are other variables involved yet unidentified in this analysis that could be explained by the hormonal changes that take place during puberty.

Physiological cardiac hypertrophy: Is the cardiac Na+/H+ exchanger (NHE1) involved? A.M. Yeves, M.B. Nolly, M.C. Villa-Abrille, O.A. Pinilla, N.G. Pérez, E.M. Escudero, I.L. Ennis Centro de Investigaciones Cardiovasculares, UNLP-CONICET, Argentina Physiological cardiac hypertrophy (CH) is an adaptive response to increased workload induced by training or pregnancy while pathological CH is due to pathological overload (i.e. hypertension) and usually evolves to arrhythmias and heart failure. We aimed to further elucidate differences in the underlying pathway in both types of CH, specially focusing in the role played by the NHE1. Physiological CH [biventricular weight/tibia length: 22.0 ± 0.3 vs. 24.3 ± 0.7 mg/mm; sedentary (S) and trained (T)] was induced in rats by swimming (90 min/day, 12 weeks). No differences in systolic function but a lower stiffness correlated with decreased collagen deposition [1.7 ± 0.05 (S) vs. 1.4 ±0.09 (N)] were detected in T. While activation of PI3-K/AKT signaling [PI3-K expression: 153 ± 18 (T) vs. 100 ± 10 (S); and P-AKT: 134 ± 10 (T) vs. 100 ± 5 (S) %]; was detected in the hypertrophied myocardium neither NHE1 [100 ± 8.5 (S) vs. 95 ± 6.7 % (N)] nor BNP (100 ± 15 vs. 99 ± 15 %) expression was modified. Similar results were obtained in isolated cardiomyocytes exposed to 10 nM IGF1. IGF1 increased myocyte area (111 ± 2 vs. 100 ± 2 %) and protein/DNA (109 ± 1.8 vs. 100 ± 1.5 %), effects not abolished by NHE1 inhibition. IGF1 also increased P-AKT (100 ± 5.9 vs. 122 ± 5.7; control and IGF1) but did not affect NHE1 activity (JH + at pHi 6.8: 2.1 ± 0.2 vs. 1.8 ± 0.2; control and IGF1). These results suggest that the NHE1; a critical mediator of pathological CH, is not involved in physiological CH.

Spironolactone inhibits the activity of Na+/H+ exchanger-1 in the aorta of DOCA-salt rats F.J. Verdugo, F.A. Montellano, F. Contreras, V. Madariaga, J.E. Carreño Laboratorio de Fisiología Integrativa y Molecular, Universidad de los Andes, Santiago, Chile

Aldosterone is known to modulate vascular tone and changes the electrolyte composition of the arterial wall, processes in which sodium transporters play an important role. Recent in vitro studies have reported that aldosterone induces Na+/H+ exchanger-1 (NHE1) expression in vascular smooth muscle cells. Aim: Clarify whether aldosterone exerts a regulation over the expression and/or activity of NHE1 in the vascular wall of desoxycorticosterone acetate (DOCA)-salt hypertensive rats. Method: Male Sprague–Dawley rats underwent unilateral nephrectomy and received DOCA 10 mg/week SC (DOCA), DOCA plus Spironolactone 25 mg/kg (DOCA + Sp), or no additional treatment (Sham) (n = 20 per group). After 4 weeks of treatment, the aorta was extracted for RT-PCR, NHE-1 activity assay through epifluorescence, and vascular contractility assay with and without amiloride (NHE1 inhibitor). Results: DOCA group presented an increased activity of NHE1 compared to Sham and DOCA + Sp (p b 0.05). There were no significant differences of NHE1 mRNA expression between groups. A decrease in arterial tension curves was observed in the DOCA group in presence of amiloride (p b 0.05); no differences were observed in the other groups. Conclusion: Aldosterone induces an increased activity of NHE1 over the vascular wall of DOCA rats. This effect is countered by Spironolactone.

High altitude (HA) arrhythmia: Is a reperfusion component involved? C. Behna,b, G.A. Dinamarcab, R.F. Jiménezc, N. De Gregorioa, V. Lipsa, D. Sozad, M. Guerrab, J. Silvae a Fac Medicina, Universidad de Chile, Chile b Mutual de Seguridad CChC, Chile c Fac Cs Físicas y Matemáticas, Universidad de Concepción, Chile d Proyecto ALMA, Chile e Fac Cs Salud, Universidad de Antofagasta, Chile HA affects biological rhythms, an ischemia–reperfusion injury possibly being involved. Heart rhythm responses to sojourning along a 25 km road between 2950 and 5050 m asl, were analyzed in male heavy truck drivers (n = 34) working at ALMA Project, an HA radiotelescope in Northern Chile. Electrocardiographic recordings (n = 46) were obtained by a portable device (holter), on ascend (mean 41 min) followed by descend (mean 38 min). Based on the difference between two consecutive RR intervals exceeding 0.16 s, 605 arrhythmic events (AE) were found. 42% and 58% of AE, respectively, evidenced on ascend and descend. RR interval duration (mean ± SD) along the 12 beats immediately preceding and following an AE on descend (0.76 ± 0.02 vs. 0.82 ± 0.06 s) differed from corresponding values (0.80 ± vs. 0.86 ± 0.06 s) on ascend (p b 0.05). High incidence of AE on descend, as well as, distinct characteristics of their beat-to-beat microenvironment, suggest a possible role of reoxygenation (and potentially of concurrent oxidative stress) in the generation of HA-related cardiovascular events. Support by FONDECYT Project No. 1100161 and facilities provided by ALMA Project are gratefully acknowledged.

Insulin and its cardioprotective effect in rat heart in ex vivo perfusion model with low-flow ischemia N. Montta, G. Sánchezb, L. Garcíaa a Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile b Facultad de Medicina, Universidad de Chile, Chile

Abstracts

Cardiovascular diseases are the most important cause of death and morbidity worldwide. In most cases ischemic events will occur, which trigger cell death mechanisms. There is evidence that insulin exerts cardioprotective effects by inhibiting apoptosis, increasing influx of glucose into the cell and stimulating cell growth and protein synthesis. The aim of this study was to evaluate whether insulin has cardioprotective effects in rat heart in an ex vivo perfusion model with low-flow ischemia. Male rats (250–300 g) were anesthetized and hearts were removed by thoracotomy and connected to a Langendorff system of retroperfusion. The hearts were subjected to different conditions of low-flow ischemia (0.25 mL/min) / reperfusion (10–14 mL/min) using a Krebs–Henseleit buffer with 11 mM glucose. Conditions were: control group, groups treated with 10 nM insulin or 100 nM only in low-flow ischemia and groups treated with 10 or 100 nM insulin during low-flow ischemia and reperfusion. For each group, average infarct size and functional parameters such as heart rate, intraventricular pressure, among others, were determined. The administration of 100 nM insulin at low-flow ischemia and 10 nM insulin during ischemia and reperfusion protected from death and significantly decreased infarct size compared to the control group (35.4 ± 32.9% v/s 45.4 ± 25.3%, and 21.4 ± 10.5% v/s 45.4 ± 25.3%, respectively). Studies using these two models will allow us to further investigate the molecular mechanisms involved in insulin-mediated cardioprotection. Fondecyt No.1110346

Ischemic postconditioning: Role of connexin 43 E.R. Diez, A. Rodríguez-Sinovas, J.A. Sánchez, A.Z. Ponce Zumino, D. García-Dorado Instituto de Fisiología, Mendoza, Argentina Vall d'Hebron Institut de Recerca, Barcelona, Spain Ischemic postconditioning (IP) reduces infarct size and reperfusion arrhythmias. To define the role of connexin 43 (Cx43) in IP protection, we analyzed myocardial electrical resistivity during reperfusion and the influence of Cx43 deficiency on these effects. IP (6 cycles of 10 s reperfusion/ischemia) reduced infarct size in isolated mice hearts from wild-type animals after 30 min of global ischemia followed by reperfusion (n = 7–9/group, p b 0.01). Tissue resistivity, determined by four electrode-probes (alternating current 10 μA, 7 kHz), was markedly increased during ischemia in control wild-type hearts, and quickly recovered during reperfusion. Interestingly, IP delayed tissue resistivity recovery by about 2 min. However, IP in Cx43-deficient hearts, either animals in which Cx43 is replaced by Cx32, or from Cx43Cre-ER(T)/fl animals, in which Cx43 expression is markedly reduced after 4-hydroxy-tamoxifen administration, was protective against infarction, and still induced a delayed and slowed recovery in tissue resistivity. We conclude that IP delays the recovery of passive myocardial electrical properties during reperfusion. However, this effect of IP, as well as its protective effect, is independent of Cx43 expression. A role for gap junctional communication in the antiarrhythmic effect of ischemic postconditioning seems unlikely.

Insulin-increased human equilibrative nucleoside transporter 2 involves Srebp-1C increased expression in human umbilical vein endothelial cells F. Pardo, F. Westermeier, C. Salomón, E. Guzmán-Gutiérrez, P. Arroyo, A. Leiva, L. Sobrevia Cellular and Molecular Physiology Laboratory, Division of Obstetrics and Gynecology, Faculty of Medicine, Pontificia Universidad Católica de Chile, Chile

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Human umbilical vein endothelial cells (HUVEC) take up adenosine via equilibrative nucleoside transporters 1 (hENT1) and hENT2. Insulin increases hENT2 expression and activity in HUVEC via unknown mechanisms. Since sterol regulatory element-binding protein-1c (Srebp-1c) mediates insulin effect in other cell types, we studied whether insulin effect on hENT2 expression involves SLC29A2 promoter activity. SLC29A2 promoter constructs were generated (pGL3-hENT2 − 1998, −1491, − 1086, −864, −602 bp from ATG) including − 1129CbA mutation (−1491mut) for Srebp binding site. HUVEC where exposed to insulin (1 nM, 8 h) and luciferase reporter activity was measured. Srebp-1c expression was determined. Insulin increased (~ 1.2 fold) the − 1491 and − 1998 constructs promoter activity, but did not alter −1491mut construct activity. Insulin increased Srebp-1c protein abundance and translocation to the nucleus (at 8 h) and mRNA expression (30 min). Insulin-increased SLC29A2 activity requires the Srebp binding consensus sequence in HUVEC. Support: CONICYT (ACT-73 PIA, AT-24120944, AT-24120941, AT-24120940), FONDECYT (1110977, 3130583, 11110059), CONICYT-PhD (EG-G, PA, CS), Faculty of Medicine, PUC-PhD (PA, CS) fellowships.

Insulin requires A2A and A2B adenosine receptors to reverse gestational diabetes reduced L-arginine transport in human placental microvascular endothelial cells E. Palacios, E. Guzmán-Gutiérrez, F. Westermeier, C. Salomón, P. Arroyo, F. Pardo, A. Leiva, L. Sobrevia Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Chile Gestational diabetes (GD) increases L-arginine transport via a mechanism involving A2A adenosine receptor (A2AAR) in human umbilical vein endothelial cells (HUVEC). However, the role of these receptors is unknown in human placental microvascular endothelial cells (hPMEC). We here evaluated whether L-arginine transport is altered by GD and whether insulin, A2AAR and A2BAR modulate GD effect in hPMEC. L-Arginine transport (0–1000 μM, 3 μCi/ml, 37 ºC, 1 min) was measured in cells from normal (Nc) or GD (GDc) pregnancies. Cells were preincubated (8 h) with insulin (1 nM) ± MRS1754 (10 nM, A2BAR antagonist) or ±ZM-241385 (10 nM, A2AAR antagonist). Maximal capacities (Vmax/Km) for L-arginine transport decreased (~90%) in GDc compared with Nc due to a lower maximal velocity, without significant alterations in the apparent Km values. LArginine transport decreased (~85%) in Nc in the presence of MRS1754 and/or ZM-241385; however, these molecules did not alter GDc effect on transport. Insulin reduced the Vmax/Km for L-arginine transport in Nc (~67%) and GDc (~95%). In presence of insulin, MRS-1754 and/or ZM-241385 decreased to ~ 90% in Nc, but in GDc with MRS-1754 or ZM-241385 increased L-arginine transport (~ 5 fold). Interestingly, coincubation of insulin + MRS-1754 + ZM241385, increased L-arginine transport (13 fold) in GDc. In conclusion, A2AAR and A2BAR could be involved in the modulation of Larginine transport in hPMEC from GD. Support: CONICYT (ACT-73 PIA, AT-24120944, AT-24120941, AT-24120940), FONDECYT (1110977, 11110059, 3130583), CONICYT-PhD fellowships (EG-G, CS, PA), PUC-Faculty of Medicine PhD fellowship (PA, CS), Chile.

Insulin reverses gestational diabetes-increased L-arginine transport involving A1 and A2A adenosine receptors activation in HUVEC E. Guzmán-Gutiérrez, C. Salomón, F. Pardo, A. Leiva, L. Sobrevia Cellular and Molecular Physiology Laboratory, Division of Obstetrics and

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Abstracts

Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Chile Gestational diabetes (GD) associates with higher expression and activity of the hCAT1 in HUVEC, a phenomenon involving A2AAR, but not A1AR activation, and increased hCAT-1-mediated L-arginine transport. We evaluated whether insulin reverses GD-increased Larginine transport and hCAT-1 protein abundance involving A1AR and A2AAR. L-Arginine transport (0–1000 μM, 3 μCi/ml, 37 °C, 1 min) and hCAT-1 protein abundance was measured in HUVEC from normal (Nc) or GD (GDc) pregnancies. Cells were preincubated with insulin (1 nM) ± DPCPX (A1AR antagonist) or ±ZM-241385 (A2AAR antagonist). GD associates with higher maximal velocity for L-arginine transport and hCAT-1 protein abundance. Insulin blocked these alterations in GDc, but increased L-arginine transport and hCAT-1 protein abundance in Nc. ZM-241385, but not DPCPX, also blocked the GD and insulin effect in Nc on L-arginine transport and hCAT-1 protein abundance. In GDc insulin effect on L-arginine transport and hCAT-1 protein abundance was blocked by DPCPX and ZM-241385. Conclusion: Insulin could be a factor reversing a GD to a normal endothelium requiring A1AR and A2AAR activation. CONICYT (ACT-73 PIA/AT-24120944/AT24120941), FONDECYT (1110977/11110059/ 3130583). CONICYT-PhD Fellowship (EG-G, CS).

Maternal obesity is associated to reduced phosphorylation of AKT and p44/42MAPK in human umbilical vein endothelial cells F. Westermeier, J.P. Kusanovic, J. Poblete, F. Mardones-Santander, L. Sobrevia, M. Farías Pontificia Universidad Católica de Chile, Santiago, Chile Maternal obesity is an independent risk factor to fetal programming of metabolic diseases, including insulin resistance (IR). Since endoplasmic reticulum (ER) stress pathway has been implicated on IR development in response to obesity, we evaluated insulin signalling response (Akt and p44/42mapk phosphorylation) and one of the ER stress markers (XBP-1 splicing) in human umbilical vein endothelial cells (HUVEC). Cells from pregnancies with maternal obesity (HUVEC-OB) and control condition (HUVEC-N) were exposed (0– 60 min) to physiological levels (1 nM) of insulin. Akt and p44/ 42mapk phosphorylation (P ~ Akt and P ~ p44/42mapk) was assayed by Western blot. XBP-1 splicing was evaluated by non-quantitative RT-PCR. HUVEC-N exposed to insulin showed a rapid increase of P ~ Akt (~ 12 fold) and P ~ p44/42mapk (~20 fold), with a maximal response at 1 min. HUVEC-OB exhibit retarded and reduced P ~ Akt (~5 fold) and p44/42mapk (~ 8 fold) in response to insulin, with a maximal increase at 15 min. Preliminary results showed no differences in XBP-1 splicing between compared groups. Altered phosphorylation levels and time-course of intracellular signalling pathways response to insulin stimulation could be associated with potential IR state in HUVEC-OB compared to HUVEC-N. FONDECYT (1121145, 1110977, 1090594), CONICYT (ACT-73 PIA).

Reduced adenosine uptake via hENT1 in human umbilical vein endothelial cells from pregnancies with pre-eclampsia M. Santos, E. Guzmán-Gutiérrez, P. Arroyo, F. Pardo, A. Leiva, L. Sobrevia Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile

Pre-eclampsia (PE) is associated with elevated maternal blood pressure, proteinuria, placental endothelial damage and dysfunction. Adenosine is a vasodilator in the human placenta vasculature. PE courses with higher plasma adenosine concentration in the umbilical vein blood, which could be due to lower adenosine transport via human equilibrative nucleoside transporters 1 (hENT1) and hENT2 in human umbilical vein endothelial cells (HUVEC). We studied whether hENT1/hENT2 adenosine uptake is altered in HUVEC from PE. Methods: Primary cultured HUVEC (passage 3) from full-term normal (N) (n = 3) and PE (n = 2) pregnancies were used for adenosine uptake (10 μM, 2 μCi/ml, 20 s, 22 °C) in the absence or presence (30 min) of nitrobenzylthioinosine (NBTI, 1 or 10 μM, hENTs inhibitor). Results: Overall adenosine uptake was decreased (~86%) in PE compared with N pregnancies. The relative hENT1 contribution to overall adenosine uptake in N pregnancies (~ 80%) was abolished in PE. However, the relative contribution of hENT2 (~20%) to overall uptake in N pregnancies was increased in PE (~ 5.5-fold). Conclusions: PE associates with reduced overall adenosine uptake most likely due to reduced hENT1 transport capacity, where altered hENT2 transport capacity may not play a significant role in the overall adenosine uptake in HUVEC. Support: CONICYT (ACT-73 PIA, AT-24120944-ATEGG y PA), FONDECYT (1110977, 11110059, 3130583), and CONICYTPhD fellowship (EG-G, PA).

Adenosine increases insulin vasodilation in human umbilical vein rings from pregnancies with preeclampsia K. Bugueñoa, M.A. Ramírezb, E. Guzmán-Gutiérreza, F. Pardoa, A. Leivaa, L. Sobreviaa a Cellular and Molecular Physiology Laboratory, Division of Obstetrics and Gynaecology, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile b Cellular Physiology Laboratory, Biomedical Department, Faculty of Health Sciences, Universidad de Antofagasta, Chile Preeclampsia (PE) is a syndrome characterized by elevated maternal blood pressure and proteinuria during pregnancy. Adenosine (Ado), acts as vasodilator in the umbilical vein (UV). We determined the effect of insulin and Ado in the vascular reactivity of human UV from normal and PE pregnancies. UV rings mounted in a myograph were pre-constricted with KCl (32.5 mM) and vasodilatation by insulin (0.1–100 nM) and Ado (1 mM) was assayed. UV rings were also preincubated (10 min) with ZM-241385 (10 nM, A2A Ado receptors (A2AAR) inhibitor). Maximal relaxation was expressed as percentage of relaxation pre-constricted with KCl. Insulin caused comparable maximal relaxations in normal and PE UV rings (~20%, IC50 10.1 ± 0.04 and 12.2 ± 0.04 nM, respectively). Insulin + Ado did not alter insulin-relaxation in normal UV; however, a higher maximal relaxation was found in PE (~ 50%, IC50 32.9 ± 0.05 nM), an effect blocked by ZM (~ 15% maximal relaxation, IC50 12.1 ± 0.07 nM). Insulin acts as vasodilator in normal and PE UV, a phenomenon potentiated by Ado only in PE. Support: CONICYT (ACT-73 PIA, AT24120944), FONDECYT (1110977, 11110059, 3130583), CONICYT-PhD fellowship (EG-G) and VRA-2009 Universidad de Antofagasta (Chile).

Insulin increases sodium-dependent L-carnitine transport involving p42/44MAPK activity in human umbilical vein endothelial cells R. Salsosoa,b, E. Guzmán-Gutiérreza, M.V. Ruiz-Armentab, S. Zambranob, C. Salomóna, P. Arroyoa, A. Blancab, F. Pardoa, A. Leivaa, A. Mateb, C. Vázquezb, L. Sobreviaa

Abstracts a

Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Chile b Department of Physiology and Zoology, Faculty of Pharmacy, Universidad de Sevilla, Seville, Spain Insulin causes vasodilation via PI3K and p42/44mapk activation in different tissues including human umbilical vein endothelium (HUVEC). L-Carnitine (LC) is a vasodilator in hypertensive rats, but its potential role on endothelial cell function is unknown. We studied membrane transport of this amino acid and evaluated whether insulin alters sodium (Na+)-dependent LC transport involving PI3K and p42/44mapk. LC transport (5–80 μM, 5 μCi/ml, 37 °C, 1 min) was measured in the absence or presence of extracellular Na+ in HUVEC from normal pregnancies. Cells were coincubated (2 h) with insulin (1 nM), wortmannin (W30 nM, PI3K inhibitor) or PD-98059 (10 μM, p42/44mapk inhibitor). LC transport was mediated by Na+-dependent

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(46% of total) and Na+-independent mechanisms (54% of total). Insulin increased the maximal velocity (Vmax) of the Na+-dependent LC transport (2.6 folds), an effect blocked by PD-98059, but unaltered by W. However, these inhibitors did not alter the Na+-dependent transport in the absence of insulin. Na+-independent LC transport was unaltered under this condition. These results suggest that insulin selectively modulates Na+-dependent LC transport likely involving p42/44mapk activity in HUVEC. Thus, a potential role for insulin as a vasodilator acting via modulation of LC uptake is proposed in HUVEC. CONICYT (ACT-73 PIA, AT-24120944, AT-24120941, AT-24120940), FONDECYT (1110977, 11110059, 3130583), CONICYT-PhD fellowships (EG-G, CS, PA), PUC-Faculty of Medicine PhD fellowship (PA, CS), Chile. Instituto de Salud Carlos III, Subdirección General de Evaluación y Fomento de la Investigación, PN de I+D+I 2008–2011 (PS09/01395) and AECID A1/036123/11 (Spain).