Mecanismo del daño mecánico letal por reoxigenación en miocitos cardíacos

Consultable des del TDXTítol obtingut de la portada digitalitzadaLos miocitos cardíacos sometidos a hipoxia prolongada presentan un gran aumento en la concentración de Ca2+ citosólico. Durante la reoxigenación, la reactivación de la producción energética mitocondrial en presencia de Ca2+ citosólico elevado provoca una activación exagerada de la contractilidad celular que puede llegar a deformar irreversiblemente el citoesqueleto y romper la membrana celular. Este fenómeno, conocido como hipercontractura, es el responsable de la muerte celular que se produce en los primeros minutos de reoxigenación y da lugar al patrón histológico de necrosis en bandas de contracción, que constituye el 90% de la necrosis en los infartos post-reperfusión. Sin embargo, en el modelo de miocitos aislados, la hipercontractura no se acompaña de muerte celular y las células hipercontraídas conservan la integridad de su sarcolema y cierta competencia metabólica. Esta discrepancia podría deberse a la ausencia de sobrecarga osmótica en el modelo de miocitos aislados. Durante la reperfusión se produce un gradiente osmótico entre el interior de las células (por los catabolitos que se han acumulado durante la isquemia) y el espacio extracelular. Como consecuencia de este gradiente entra agua al interior de las células creando un aumento de la tensión sobre la membrana que puede llegar a romperla. En este estudio se ha investigado la contribución del edema sobre la viabilidad celular en miocitos aislados sometidos a anoxia simulada y reoxigenación hipoosmótica, el papel de la fragilidad estructural, su evolución temporal y los mecanismos responsables de la misma. Además, se ha estudiado la interacción celular como posible mecanismo adicional de daño, utilizando parejas de miocitos conectados y sometidos a técnicas de micromanipulación. Los resultados demuestran que la hipercontractura coopera con la sobrecarga osmótica en la rotura de la membrana en miocitos reoxigenados. En cambio, el edema y la hipercontractura no producen disrupción del sarcolema en miocitos que no han estado sometidos a un período previo de anoxia. Así pues, la sobrecarga mecánica impuesta por el edema y la hipercontractura producen la muerte celular por rotura del sarcolema en miocitos que han desarrollado fragilidad estructural secundaria a la deprivación energética. La fragilidad no se recupera durante los primeros 40min de reoxigenación. La preincubación de los miocitos con TMZ, un fármaco estabilizante de los lípidos de membrana, es capaz de aumentar la resistencia mecánica y mejorar la supervivencia de células reoxigenadas sometidas a estrés osmótico. Este efecto de la TMZ indica que la fragilidad osmótica de los miocitos reoxigenados está relacionada, al menos en parte, con un proceso de degradación lipídica del sarcolema durante la anoxia. Además, los fármacos donadores de NO y antioxidantes lipídicos son capaces de mejorar la viabilidad celular en miocitos sometidos a estrés osmótico. El mecanismo de acción de estos fármacos no se conoce, aunque parece ser independiente de la vía del GMPc, lo que es compatible con la hipótesis de que el incremento de la fragilidad observada durante la reoxigenación se debe en parte a la acción de los radicales libres del oxígeno. Finalmente, en parejas de miocitos unidos por discos intercalares intactos, la hipercontractura de un miocito es capaz de transmitirse a un miocito adyacente. La transmisión célula a célula de la hipercontractura se puede prevenir utilizando heptanol, un bloqueante de los gap junctions. Estos resultados apoyan la hipótesis de que la interacción química a través de los gap junctions entre células adyacentes puede contribuir a la transmisión de la hipercontractura. En conclusión, los resultados de este estudio demuestran la existencia de diferentes mecanismos capaces de causar la muerte celular durante los primeros minutos de reperfusión, y abren la posibilidad de prevenirla mediante intervenciones farmacológicas aplicadas en el momento de la reinstauración del flujo coronario.Prolonged hypoxia induces cytosolic Ca2+ overload in cardiac myocytes. During reoxygenation, reactivation of mitochondrial ATP production in the presence of high cytosolic Ca2+ levels induces an exaggerated contractile activity that eventually leads to irreversible cytoskeleton as well as sarcolemmal disruption. This phenomenon, also known as hypercontracture, is the main mechanism of the cell death taking place in the first minutes of reoxygenation, and gives rise to the characteristic histological pattern of contraction band necrosis present in the 90% of the post-reperfused necrotic myocardium. Nevertheless, hypercontracture is not associated to cell death in isolated myocytes, and hypercontracted cells maintain their sarcolemmal integrity and metabolic competence. This discrepancy could be due to the lack of osmotic stress in the isolated myocyte model. During reperfusion, an important osmotic gradient develops between the cells (which contain many catabolites accumulated during the previous ischemic period) and the extracellular space. As a consequence, water goes into the cells causing an increase in the membrane tension that eventually could induce its disruption. This study has investigated the contribution of swelling on cell viability in isolated myocytes submitted to simulated anoxia and hypoosmotic reoxygenation, the role of structural fragility, its temporal progression and the mechanisms involved in it. Moreover, cell interaction has been analysed as a potential mechanism of cell injury, by means of pairs of connected myocytes subjected to micromanipulation techniques. The results demonstrate a cooperation between hypercontracture and osmotic swelling in inducing membrane rupture of reoxygenated myocytes. However, swelling and hypercontracture does not promote sarcolemmal disruption when myocytes have not been subjected to a previous anoxic period. Thus, mechanical stress secondary to cell swelling and hypercontracture induces sarcolemmal disruption and cell death in myocytes with previous structural fragility secondary to energy deprivation. Fragility is not recovered at least during the first 40min of reoxygenation. Preincubation of myocytes with TMZ, a drug with lipid membrane stabilizing properties, is associated to an increase in mechanical resistance and to a better cell survival during reoxygenation in the presence of osmotic stress. This effect suggests that osmotic fragility in reoxygenated myocytes is related, at least in part, with sarcolemmal lipid degradation during anoxia. Moreover, NO donors and lipid antioxidants improve cell viability in myocytes submitted to osmotic stress. The mechanism of action of these drugs is not known, though it seems to be independent of GMPc pathway. This is in agreement with the hypothesis of the role of oxygen free radicals in the induction of reoxygenation-induced fragility. In pairs of connected myocytes through intact intercalated disks, hypercontracture of one of the cells can be transmitted to the neighboring cell. This cell-to-cell transmission of hypercontracture can be inhibited by heptanol, a gap junction blocker. These results suggest that chemical interaction through gap junctions between adjacent cells may contribute to transmission of hypercontracture. In conclusion, the results of this study demonstrate for the first time the cooperation of several different mechanisms to induce cell death during the very first minutes of reperfusion. The identification of these mechanisms opens the possibility of developing novel therapeutical approaches addressed to reduce cell death in the early phase of coronary flow restoration

Connexin 43 in Mitochondria: What Do We Really Know About Its Function?

Connexin: Ischemia-reperfusion injury; MitochondriaConexina; Lesión por isquemia-reperfusión; MitocondriasConnexina; Lesió per isquèmia-reperfusió; MitocondrisConnexins are known for their ability to mediate cell-cell communication via gap junctions and also form hemichannels that pass ions and molecules over the plasma membrane when open. Connexins have also been detected within mitochondria, with mitochondrial connexin 43 (Cx43) being the best studied to date. In this review, we discuss evidence for Cx43 presence in mitochondria of cell lines, primary cells and organs and summarize data on its localization, import and phosphorylation status. We further highlight the influence of Cx43 on mitochondrial function in terms of respiration, opening of the mitochondrial permeability transition pore and formation of reactive oxygen species, and also address the presence of a truncated form of Cx43 termed Gja1-20k. Finally, the role of mitochondrial Cx43 in pathological conditions, particularly in the heart, is discussed.KB is funded by the German Research Foundation (BO 2955/4-1). LL is supported by the Research Foundation Flanders (FWO) grant numbers G.0527.18N and G040720N. MR-M is supported by the Instituto de Salud Carlos III of the Spanish Ministry of Health (FIS-PI19-01196) and a grant from the Sociedad Española de Cardiología (SEC/FEC-INV- BAS 217003)

Dicarbonyl stress and mitochondrial dysfunction in the aged heart

Aging; Cardiomyocytes; MitochondriaEnvejecimiento; Cardiomiocitos; MitocondriasEnvelliment; Cardiomiòcits; Mitocondri

A novel double-hit animal model of schizophrenia: behavioural assessment in male and female mice

Background: A growing body of evidence support that maternal prenatal infections represent a risk factor for schizophrenia in offspring. Moreover, stressful events during critical neurodevelopmental periods, such as adolescence, may trigger the onset of the disease in predisposed individuals. Thus, a prenatal priming event (i.e. maternal infection during pregnancy) that would induce vulnerability, followed by a second stressful hit in peripuberty may lead to the onset of schizophreniaDisclosure statement: Supported by the Basque Government (IT1211-19), the European Union's Horizon 2020 research and innovation programme (Marie Sklodowska-Curie grant agreement No 747487) and SAF 2017-88126-R. NC is recipient of a FPI fellowship (PRE2018-084002) from MICINN

IMproving Preclinical Assessment of Cardioprotective Therapies (IMPACT) criteria: guidelines of the EU-CARDIOPROTECTION COST Action

Cardioprotection; Drug development; InfarctionCardioprotección; Desarrollo de fármacos; InfartoCardioprotecció; Desenvolupament de fàrmacs; InfartAcute myocardial infarction (AMI) and the heart failure (HF) which may follow are among the leading causes of death and disability worldwide. As such, new therapeutic interventions are still needed to protect the heart against acute ischemia/reperfusion injury to reduce myocardial infarct size and prevent the onset of HF in patients presenting with AMI. However, the clinical translation of cardioprotective interventions that have proven to be beneficial in preclinical animal studies, has been challenging. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic in vivo preclinical assessment of the efficacy of promising cardioprotective interventions prior to their clinical evaluation. To address this, we propose an in vivo set of step-by-step criteria for IMproving Preclinical Assessment of Cardioprotective Therapies (‘IMPACT’), for investigators to consider adopting before embarking on clinical studies, the aim of which is to improve the likelihood of translating novel cardioprotective interventions into the clinical setting for patient benefit.This article is based on the work from COST Action EU-CARDIOPROTECTION CA16225 supported by COST (European Cooperation in Science and Technology). DJH is supported by the Duke-National University Singapore Medical School, Singapore Ministry of Health’s National Medical Research Council under its Clinician Scientist-Senior Investigator scheme (NMRC/CSA-SI/0011/2017) and Collaborative Centre Grant scheme (NMRC/CGAug16C006). SL is supported by grants from the South African Department of Science and Technology and the South African National Research Foundation. SMD is supported by grants from the British Heart Foundation (PG/19/51/34493 and PG/16/85/32471). GH is supported by the German Research Foundation (SFB 1116 B8). MRM is supported by the Spanish Institute of Health Carlos III (FIS PI19/01196 and CIBER-CV). RS is supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [Project number 268555672—SFB 1213, Project B05]. PF is supported by the National Research, Development and Innovation Office of Hungary (Research Excellence Program—TKP, National Heart Program NVKP 16-1-2016-0017) and by the Higher Education Institutional Excellence Program of the Ministry of Human Capacities in Hungary, within the framework of the Therapeutic Development thematic program of the Semmelweis University

Calpains as Potential Therapeutic Targets for Myocardial Hypertrophy

Calpain; Calpastatin; Myocardial hypertrophyCalpaína; Calpastatina; Hipertrofia miocárdicaCalpaïna; Calpastatina; Hipertròfia miocàrdicaDespite advances in its treatment, heart failure remains a major cause of morbidity and mortality, evidencing an urgent need for novel mechanism-based targets and strategies. Myocardial hypertrophy, caused by a wide variety of chronic stress stimuli, represents an independent risk factor for the development of heart failure, and its prevention constitutes a clinical objective. Recent studies performed in preclinical animal models support the contribution of the Ca2+-dependent cysteine proteases calpains in regulating the hypertrophic process and highlight the feasibility of their long-term inhibition as a pharmacological strategy. In this review, we discuss the existing evidence implicating calpains in the development of cardiac hypertrophy, as well as the latest advances in unraveling the underlying mechanisms. Finally, we provide an updated overview of calpain inhibitors that have been explored in preclinical models of cardiac hypertrophy and the progress made in developing new compounds that may serve for testing the efficacy of calpain inhibition in the treatment of pathological cardiac hypertrophy.This study was funded by the Instituto de Salud Carlos III of the Spanish Ministry of Health FIS-PI20/01681) and the research network CIBERCV (CB16/11/00479)

El precondicionamiento isquémico produce cambios en las proteínas de las fosforilación oxidativa y atenúa el daño oxidativo durante la isquemia-reperfusión por mecanismos independientes de la señalización citosólica

Comunicaciones a congreso

Interaction of Cardiovascular Nonmodifiable Risk Factors, Comorbidities and Comedications With Ischemia/Reperfusion Injury and Cardioprotection by Pharmacological Treatments and Ischemic Conditioning

Risc cardiovascular; Isquèmia/reperfusióCardiovascular risk; Ischemia/reperfusionRiesgo cardiovascular; Isquemia/reperfusiónPreconditioning, postconditioning, and remote conditioning of the myocardium enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and the potential to provide novel therapeutic paradigms for cardioprotection. While many signaling pathways leading to endogenous cardioprotection have been elucidated in experimental studies over the past 30 years, no cardioprotective drug is on the market yet for that indication. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic preclinical evaluation of promising cardioprotective therapies prior to their clinical evaluation, since ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors and comorbidities. These risk factors and comorbidities induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury and responses to cardioprotective interventions. Moreover, some of the medications used to treat these comorbidities may impact on cardioprotection by again modifying cellular signaling pathways. The aim of this article is to review the recent evidence that cardiovascular risk factors as well as comorbidities and their medications may modify the response to cardioprotective interventions. We emphasize the critical need for taking into account the presence of cardiovascular risk factors as well as comorbidities and their concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple comorbidities. Significance Statement Ischemic heart disease is a major cause of mortality; however, there are still no cardioprotective drugs on the market. Most studies on cardioprotection have been undertaken in animal models of ischemia/reperfusion in the absence of comorbidities; however, ischemic heart disease develops with other systemic disorders (e.g., hypertension, hyperlipidemia, diabetes, atherosclerosis). Here we focus on the preclinical and clinical evidence showing how these comorbidities and their routine medications affect ischemia/reperfusion injury and interfere with cardioprotective strategies.P.F. was supported by the National Research, Development and Innovation Office of Hungary (Research Excellence Program–TKP, National Heart Program NVKP 16-1-2016-0017) and by the Higher Education Institutional Excellence Program of the Ministry of Human Capacities in Hungary, within the framework of the Therapeutic Development thematic program of Semmelweis University. D.D. is supported by grants from National Institutes of Health National Heart, Lung, and Blood Institute [R01-HL136389, R01-HL131517, R01-HL089598, and R01-HL163277], the German Research Foundation [DFG, Do 769/4-1], the European Union (large-scale integrative project MAESTRIA, no. 965286). G.H. is supported by the German Research Foundation [SFB 1116 B8]. D.H. is supported by the Duke–NUS Signature Research Programme funded by the Ministry of Health, Singapore Ministry of Health’s National Medical Research Council under its Clinician Scientist–Senior Investigator scheme [NMRC/CSA-SI/0011/2017], Centre Grant [CGAug16M006], and Collaborative Centre Grant scheme [NMRC/CGAug16C006]. I.A. is supported from Boehringer-Ingelheim for the investigation of the effects of empagliflozin on the myocardium and from the European Union (ERDF) and Greek national funds through the Operational Program “Competitiveness, Entrepreneurship and Innovation,” under the call “RESEARCH – CREATE – INNOVATE” (project code: 5048539). S.M.D. acknowledges the support of the British Heart Foundation [PG/19/51/34493 and PG/16/85/32471]. S.L. is supported by the South African National Research Foundation and received COST Seed funding from the Department of Science and Innovation in South Africa. M.R-M. is supported by the Instituto de Salud Carlos III of the Spanish Ministry of Health [FIS-PI19-01196] and a grant from the Spanish Society of Cardiology [SEC/FEC-INV-BAS 217003]. C.J.Z. is supported by a grant from European Foundation for the Study of Diabetes (EFSD), a research grant from Boehringer-Ingelheim and an institutional grant from Amsterdam UMC Cardiovascular Research. R.S. is supported by Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) [Project number 268555672—SFB 1213, Project B05]

Cardiac-Specific Overexpression of ERRγ in Mice Induces Severe Heart Dysfunction and Early Lethality

Disfunció cardíaca; Receptors relacionats amb estrògens; Ratolins transgènicsDisfunción cardíaca; Receptores relacionados con el estrógeno; Ratones transgénicosCardiac dysfunction; Estrogen-related receptors; Transgenic miceProper cardiac function depends on the coordinated expression of multiple gene networks related to fuel utilization and mitochondrial ATP production, heart contraction, and ion transport. Key transcriptional regulators that regulate these gene networks have been identified. Among them, estrogen-related receptors (ERRs) have emerged as crucial modulators of cardiac function by regulating cellular metabolism and contraction machinery. Consistent with this role, lack of ERRα or ERRγ results in cardiac derangements that lead to functional maladaptation in response to increased workload. Interestingly, metabolic inflexibility associated with diabetic cardiomyopathy has been recently associated with increased mitochondrial fatty acid oxidation and expression of ERRγ, suggesting that sustained expression of this nuclear receptor could result in a cardiac pathogenic outcome. Here, we describe the generation of mice with cardiac-specific overexpression of ERRγ, which die at young ages due to heart failure. ERRγ transgenic mice show signs of dilated cardiomyopathy associated with cardiomyocyte hypertrophy, increased cell death, and fibrosis. Our results suggest that ERRγ could play a role in mediating cardiac pathogenic responses.Research was funded by “Fundació la Marató de TV3” (# 082610 to JAV) and by Instituto de Salud Carlos III (PI19/00167 to MZ, co-funded by European Regional Development Fund -FEDER- “A way to make Europe”)

Therapeutic S100A8/A9 blockade inhibits myocardial and systemic inflammation and mitigates sepsis-induced myocardial dysfunction

Endotoxemia; Inflammation; Mitochondrial functionEndotoxemia; Inflamación; Función mitocondrialEndotoxèmia; Inflamació; Funció mitocondrialBackground and Aims The triggering factors of sepsis-induced myocardial dysfunction (SIMD) are poorly understood and are not addressed by current treatments. S100A8/A9 is a pro-inflammatory alarmin abundantly secreted by activated neutrophils during infection and inflammation. We investigated the efficacy of S100A8/A9 blockade as a potential new treatment in SIMD. Methods The relationship between plasma S100A8/A9 and cardiac dysfunction was assessed in a cohort of 62 patients with severe sepsis admitted to the intensive care unit of Linköping University Hospital, Sweden. We used S100A8/A9 blockade with the small-molecule inhibitor ABR-238901 and S100A9−/− mice for therapeutic and mechanistic studies on endotoxemia-induced cardiac dysfunction in mice. Results In sepsis patients, elevated plasma S100A8/A9 was associated with left-ventricular (LV) systolic dysfunction and increased SOFA score. In wild-type mice, 5 mg/kg of bacterial lipopolysaccharide (LPS) induced rapid plasma S100A8/A9 increase and acute LV dysfunction. Two ABR-238901 doses (30 mg/kg) administered intraperitoneally with a 6 h interval, starting directly after LPS or at a later time-point when LV dysfunction is fully established, efficiently prevented and reversed the phenotype, respectively. In contrast, dexamethasone did not improve cardiac function compared to PBS-treated endotoxemic controls. S100A8/A9 inhibition potently reduced systemic levels of inflammatory mediators, prevented upregulation of inflammatory genes and restored mitochondrial function in the myocardium. The S100A9−/− mice were protected against LPS-induced LV dysfunction to an extent comparable with pharmacologic S100A8/A9 blockade. The ABR-238901 treatment did not induce an additional improvement of LV function in the S100A9−/− mice, confirming target specificity. Conclusion Elevated S100A8/A9 is associated with the development of LV dysfunction in severe sepsis patients and in a mouse model of endotoxemia. Pharmacological blockade of S100A8/A9 with ABR-238901 has potent anti-inflammatory effects, mitigates myocardial dysfunction and might represent a novel therapeutic strategy for patients with severe sepsis.Open access funding provided by Lund University. This study was supported by grants from the Marianne and Marcus Wallenberg Foundation the Swedish Heart and Lung Foundation, the Swedish Research Council, the Bundy Academy foundation at Lund University, Skåne Region Research Funds, Malmö University Hospital Funds, the Crafoord Foundation, the Royal Physiographic Society in Lund, Swedish Research Council, Östergotland Region Research Funds, and the Ministry of Research and Education of Romania (PNRR-C9/I8-CF148)