RESUMEN

Many cardiac insults causing atrial remodeling are linked to either stretch or tachycardia, but a comparative characterization of their effects on early remodeling events in human myocardium is lacking. Here, we applied isometric stretch or sustained tachycardia at 2.5 Hz in human atrial trabeculae for 6 h followed by microarray gene expression profiling. Among largely independent expression patterns, we found a small common fraction with the microRNA miR-1183 as the highest up-regulated transcript (up to 4-fold). Both, acute stretch and tachycardia induced down-regulation of the predicted miR-1183 target genes ADAM20 and PLA2G7. Furthermore, miR-1183 was also significantly up-regulated in chronically remodeled atrial samples from patients with persistent atrial fibrillation (3-fold up-regulation versus sinus rhythm samples), and in ventricular myocardium from dilative cardiomyopathy hearts (2-fold up-regulation) as compared to non-failing controls. In sum, although stretch and tachycardia show distinct transcriptomic signatures in human atrial myocardium, both cardiac insults consistently regulate the expression of miR-1183 and its downstream targets in acute and chronic remodeling. Thus, elevated expression of miR-1183 might serve as a tissue biomarker for atrial remodeling and might be of potential functional significance in cardiac disease.

Asunto(s)

Fibrilación Atrial , Remodelación Atrial , MicroARNs , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Biomarcadores/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Miocardio/metabolismo , Taquicardia/genética , Taquicardia/metabolismo

RESUMEN

Background Experiments measuring the contractile properties of human myocardium are important for translational research but complicated by the logistical difficulties of acquiring specimens. Accordingly, many groups perform contractile assays using samples that are acquired from patients at one institution and shipped to another institution for experiments. This necessitates freezing the samples and performing subsequent assays using chemically permeabilized preparations. It is unknown how prior freezing affects the contractile function of these preparations. Methods and Results To examine the effects of freezing we measured the contractile function of never-frozen and previously frozen myocardial samples. Samples of left ventricular tissue were obtained from 7 patients who were having a ventricular assist device implanted. Half of each sample was chemically permeabilized and used immediately for contractile assays. The other half of the sample was snap frozen in liquid nitrogen and maintained at -180 °C for at least 6 months before being thawed and tested in a second series of experiments. Maximum isometric force measured in pCa 4.5 solution, passive force measured in pCa 9.0 solution, and Hill coefficients were not influenced by prior freezing (P=0.07, P=0.14, and P=0.27 respectively). pCa50 in never-frozen samples (6.11±0.04) was statistically greater (P<0.001) than that measured after prior freezing (5.99±0.04) but the magnitude of the effect was only ≈0.1 pCa units. Conclusions We conclude that prior freezing has minimal impact on the contractile properties that can be measured using chemically permeabilized human myocardium.

Asunto(s)

Contracción Miocárdica , Miocardio , Congelación , Ventrículos Cardíacos , Humanos

RESUMEN

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for disease modeling and drug cardiotoxicity screening. To this end, we recently developed human cardiac organoids (hCOs) for modeling human myocardium. Here, we perform a transcriptomic analysis of various in vitro hiPSC-CM platforms (2D iPSC-CM, 3D iPSC-CM and hCOs) to deduce the strengths and limitations of these in vitro models. We further compared iPSC-CM models to human myocardium samples. Our data show that the 3D in vitro environment of 3D hiPSC-CMs and hCOs stimulates the expression of genes associated with tissue formation. The hCOs demonstrated diverse physiologically relevant cellular functions compared to the hiPSC-CM only models. Including other cardiac cell types within hCOs led to more transcriptomic similarities to adult myocardium. hCOs lack matured cardiomyocytes and immune cells, which limits a complete replication of human adult myocardium. In conclusion, 3D hCOs are transcriptomically similar to myocardium, and future developments of engineered 3D cardiac models would benefit from diversifying cell populations, especially immune cells.

Asunto(s)

Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Organoides/metabolismo , Transcriptoma , Adulto , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocardio/citología , Miocitos Cardíacos/citología , Organoides/citología

RESUMEN

This study measured how heart failure affects the contractile properties of the human myocardium from the left and right ventricles. The data showed that maximum force and maximum power were reduced by approximately 30% in multicellular preparations from both ventricles, possibly because of ventricular remodeling (e.g., cellular disarray and/or excess fibrosis). Heart failure increased the calcium (Ca2+) sensitivity of contraction in both ventricles, but the effect was bigger in right ventricular samples. The changes in Ca2+ sensitivity were associated with ventricle-specific changes in the phosphorylation of troponin I, which indicated that adrenergic stimulation might induce different effects in the left and right ventricles.

RESUMEN

The adipocytokine resistin is released from epicardial adipose tissue (EAT). Plasma resistin and EAT deposition are independently associated with atrial fibrillation. The EAT secretome enhances arrhythmia susceptibility and inotropy of human myocardium. Therefore, we aimed to determine the effect of resistin on the function of human myocardium and how resistin contributes to the proarrhythmic effect of EAT. EAT biopsies were obtained from 25 cardiac surgery patients. Resistin levels were measured by ELISA in 24-h EAT culture media (n = 8). The secretome resistin concentrations increased over the culture period to a maximal level of 5.9 ± 1.2 ng/mL. Coculture with ß-adrenergic agonists isoproterenol (n = 4) and BRL37344 (n = 13) had no effect on EAT resistin release. Addition of resistin (7, 12, 20 ng/mL) did not significantly increase the spontaneous contraction propensity of human atrial trabeculae (n = 10) when given alone or in combination with isoproterenol. Resistin dose-dependently increased trabecula-developed force (maximal 2.9-fold increase, P < 0.0001), as well as the maximal rates of contraction (2.6-fold increase, P = 0.002) and relaxation (1.8-fold increase, P = 0.007). Additionally, the postrest potentiation capacity of human trabeculae was reduced at all resistin doses, suggesting that the inotropic effect induced by resistin might be due to altered sarcoplasmic reticulum Ca2+ handling. EAT resistin release is not modulated by common arrhythmia triggers. Furthermore, exogenous resistin does not promote arrhythmic behavior in human atrial trabeculae. Resistin does, however, induce an acute dose-dependent positive inotropic and lusitropic effect.

Asunto(s)

Arritmias Cardíacas/inducido químicamente , Atrios Cardíacos/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Resistina/fisiología , Tejido Adiposo/metabolismo , Anciano , Anciano de 80 o más Años , Calcio/metabolismo , Cardiotónicos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoproterenol/farmacología , Masculino , Persona de Mediana Edad , Pericardio/metabolismo , Resistina/sangre , Retículo Sarcoplasmático/metabolismo , Malla Trabecular/metabolismo

RESUMEN

Human heart samples from the Sydney Heart Bank have become a de facto standard against which others can be measured. Crucially, the heart bank contains a lot of donor heart material: for most researchers this is the hardest to obtain and yet is necessary since we can only study the pathological human heart in comparison with a control, preferably a normal heart sample. It is not generally realised how important the control is for human heart studies. We review our studies on donor heart samples. We report the results obtained with 17 different donor samples collected from 1994 to 2011 and measured from 2005 to 2015 by our standard methodology for in vitro motility and troponin I phosphorylation measurements. The donor heart sample parameters are consistent between the hearts, over time and with different operators indicating that Sydney Heart Bank donor hearts are a valid baseline control for comparison with pathological heart samples. We also discuss to what extent donor heart samples are representative of the normal heart.

RESUMEN

Experiments on animal hearts (rat, rabbit, guinea pig, etc.) have demonstrated that mechano-calcium feedback (MCF) and mechano-electric feedback (MEF) are very important for myocardial self-regulation because they adjust the cardiomyocyte contractile function to various mechanical loads and to mechanical interactions between heterogeneous myocardial segments in the ventricle walls. In in vitro experiments on these animals, MCF and MEF manifested themselves in several basic classical phenomena (e.g., load dependence, length dependence of isometric twitches, etc.), and in the respective responses of calcium transients and action potentials. However, it is extremely difficult to study simultaneously the electrical, calcium, and mechanical activities of the human heart muscle in vitro. Mathematical modeling is a useful tool for exploring these phenomena. We have developed a novel model to describe electromechanical coupling and mechano-electric feedbacks in the human cardiomyocyte. It combines the 'ten Tusscher-Panfilov' electrophysiological model of the human cardiomyocyte with our module of myocardium mechanical activity taken from the 'Ekaterinburg-Oxford' model and adjusted to human data. Using it, we simulated isometric and afterloaded twitches and effects of MCF and MEF on excitation-contraction coupling. MCF and MEF were found to affect significantly the duration of the calcium transient and action potential in the human cardiomyocyte model in response to both smaller afterloads as compared to bigger ones and various mechanical interventions applied during isometric and afterloaded twitches.

Asunto(s)

Calcio/farmacología , Simulación por Computador , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Estimulación Eléctrica , Fenómenos Electrofisiológicos , Acoplamiento Excitación-Contracción , Humanos , Potenciales de la Membrana , Contracción Miocárdica/fisiología

RESUMEN

The aim of the study was to develop a new technology for the detection of amyloid in human tissues based on the fluorescent dye, disodium salt of 2,7-(1-amino-4-sulfo-2-naphthylazo)fluorene (DSNAF). MATERIALS AND METHODS: Synthesis of DSNAF was performed by diazotization of 2,7-diaminofluorene in a stream of argon followed by azo coupling with naphthionic acid. Identification of DSNAF was performed using MALDI mass spectrometry. Human myocardial samples from males and females aged from 85 to 98 years (n=11) were the material for the histochemical study. Myocardial paraffin sections were stained with a 0.1% aqueous solution of Congo red or with an aqueous solution (0.1 or 0.034%) of DSNAF under the same conditions. RESULTS: It has been demonstrated for the first time that a new fluorene-based analogue of Congo red, DSNAF, can be successfully used to identify amyloid deposits in histological sections of human myocardium. In terms of the specificity and intensity of amyloid staining, DSNAF is comparable to Congo red, which is the gold standard for detecting amyloid deposits. The fluorescence intensity of DSNAF when binding to amyloid fibrils is significantly higher than the intensity of Congo red fluorescence (with a lower intensity of background fluorescence of heart muscle tissue). This is especially useful for identifying small deposits of amyloid in the human tissues which is important when using small biopsies. CONCLUSION: The advantages of using DSNAF allow us to consider the developed technology for the detection of amyloid as a new promising method of identifying amyloid deposits in human tissues.

RESUMEN

Redox signaling affects all aspects of cardiac function and homeostasis. With the development of genetically encoded fluorescent redox sensors, novel tools for the optogenetic investigation of redox signaling have emerged. Here, we sought to develop a human heart muscle model for in-tissue imaging of redox alterations. For this, we made use of (1) the genetically-encoded Grx1-roGFP2 sensor, which reports changes in cellular glutathione redox status (GSH/GSSG), (2) human embryonic stem cells (HES2), and (3) the engineered heart muscle (EHM) technology. We first generated HES2 lines expressing Grx1-roGFP2 in cytosol or mitochondria compartments by TALEN-guided genomic integration. Grx1-roGFP2 sensor localization and function was verified by fluorescence imaging. Grx1-roGFP2 HES2 were then subjected to directed differentiation to obtain high purity cardiomyocyte populations. Despite being able to report glutathione redox potential from cytosol and mitochondria, we observed dysfunctional sarcomerogenesis in Grx1-roGFP2 expressing cardiomyocytes. Conversely, lentiviral transduction of Grx1-roGFP2 in already differentiated HES2-cardiomyocytes and human foreskin fibroblast was possible, without compromising cell function as determined in EHM from defined Grx1-roGFP2-expressing cardiomyocyte and fibroblast populations. Finally, cell-type specific GSH/GSSG imaging was demonstrated in EHM. Collectively, our observations suggests a crucial role for redox signaling in cardiomyocyte differentiation and provide a solution as to how this apparent limitation can be overcome to enable cell-type specific GSH/GSSG imaging in a human heart muscle context.

RESUMEN

BACKGROUND: Left ventricular hypertrophy and myocardial remodeling occur with aortic valve disease and may lead to heart failure. Although increased oxidative stress and inflammatory factors have been implicated in heart failure, their role in the progression of valve disease remains unclear. OBJECTIVES: We investigated the role of oxidative stress and inflammatory factors in valve disease whether this relates to cell death. METHODS: Blood samples were taken from 24 patients with valve disease before surgery and the results were compared with those from blood samples from 30 control healthy subjects. Myocardial biopsies from patients with valve disease were also collected before cannulation of the right atrial appendage. NF-κB activities in atrial and mononuclear cells nuclear extracts were determined by electrophoretic mobility shift assay. RESULTS: Nuclear factor kappaB activities were significantly greater in mononuclear cells from AVD patients compared with healthy controls and the antigens were detectable in atrial tissues valve disease patients. Plasma C-reactive protein, B-natriuretic peptides, plasma tumor necrosis factor alpha and soluble tumor necrosis factor receptor 1 and 3-nitrotyrosine levels were significantly higher in valve disease patients. Inducible nitric oxide and 3-nitrotyrosine antigens and cells expressing CD45 antigens were detected within atrial tissues obtained from valve disease patients suggesting oxidative stress originated from in situ leukocytes. CONCLUSION: The findings suggest that oxidative stress originating from in situ leukocytes within the atrial myocardium may be the potential trigger for excessive transcriptional activities and apoptotic cell death within the atrial myocardium of valve disease patients. This represents a potential therapeutic target.

Asunto(s)

Muerte Celular/fisiología , Cardiopatías Congénitas/fisiopatología , Enfermedades de las Válvulas Cardíacas/fisiopatología , Miocardio/patología , Válvula Aórtica/fisiopatología , Enfermedad de la Válvula Aórtica Bicúspide , Femenino , Humanos , Masculino , Estrés Oxidativo

RESUMEN

We aimed to identify and quantify CD117+ and CD90+ endogenous cardiac progenitor cells (CPC) in human healthy and diseased hearts. We hypothesize that these cells perform a locally acting, contributing function in overcoming medical conditions of the heart by endogenous means. Human myocardium biopsies were obtained from 23 patients with the following diagnoses: Dilatative cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), myocarditis, and controls from healthy cardiac patients. High-resolution scanning microscopy of the whole slide enabled a computer-based immunohistochemical quantification of CD117 and CD90. Those signals were evaluated by Definiens Tissue Phenomics® Technology. Co-localization of CD117 and CD90 was determined by analyzing comparable serial sections. CD117+/CD90+ cardiac cells were detected in all biopsies. The highest expression of CD90 was revealed in the myocarditis group. CD117 was significantly higher in all patient groups, compared to healthy specimens (*p < 0.05). The highest co-expression was found in the myocarditis group (6.75 ± 3.25 CD90+CD117+ cells/mm2) followed by ICM (4 ± 1.89 cells/mm2), DCM (1.67 ± 0.58 cells/mm2), and healthy specimens (1 ± 0.43 cells/mm2). We conclude that the human heart comprises a fraction of local CD117+ and CD90+ cells. We hypothesize that these cells are part of local endogenous progenitor cells due to the co-expression of CD90 and CD117. With novel digital image analysis technologies, a quantification of the CD117 and CD90 signals is available. Our experiments reveal an increase of CD117 and CD90 in patients with myocarditis.

RESUMEN

Cardiac tissue engineering by means of synthetic or natural scaffolds combined with stem/progenitor cells is emerging as the response to the unsatisfactory outcome of approaches based solely on the injection of cells. Parenchymal and supporting cells are surrounded, in vivo, by a specialized and tissue-specific microenvironment, consisting mainly of extracellular matrix (ECM) and soluble factors incorporated in the ECM. Since the naturally occurring ECM is the ideal platform for ensuring cell engraftment, survival, proliferation, and differentiation, the acellular native ECM appears by far the most promising and appealing substrate among all biomaterials tested so far. To obtain intact scaffold of human native cardiac ECM while preserving its composition, we compared the decellularized ECM (d-ECM) produced through five different protocols of decellularization (named Pr1, Pr2, Pr3, Pr4, and Pr5) in terms of efficiency of decellularization, composition, and three-dimensional architecture of d-ECM scaffolds and of their suitability for cell repopulation. The decellularization procedures proved substantially different. Specifically, only three, of the five protocols tested, proved effective in producing thoroughly acellular d-ECM. In addition, the d-ECM delivered differed in architecture and composition and, more importantly, in its ability to support engraftment, survival, and differentiation of cardiac primitive cells in vitro.

Asunto(s)

Miocardio/citología , Prótesis e Implantes , Ingeniería de Tejidos/métodos , Colágeno/metabolismo , ADN/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Persona de Mediana Edad

RESUMEN

The ERBB2 gene encodes a transmembrane tyrosine kinase receptor that belongs to the epidermal growth factor receptor (EGFR) family. ERBB2 plays a pivotal role during heart development and is essential for normal cardiac function, particularly during episodes of cardiac stress. The monoclonal antibody drug trastuzumab is used for the therapy of breast cancers that overexpress ERBB2. The clinical use of trastuzumab is limited by the development of cardiotoxicity in some patients. Inter-individual differences in the expression of ERBB2 in cardiac tissue may impact the risk of cardiotoxicity. In this study, we examined whether DNA methylation status in the proximal promoter region of ERBB2 is associated to variable ERBB2 mRNA and ERBB2 protein expression in human myocardium. Complementary studies with ERBB2 gene reporter constructs and chromatin immunoprecipitation suggest that differential methylation in specific CpG sites modify the binding of Sp1 to the promoter of ERBB2. DNA methylation in the ERBB2 locus may contribute to the variable expression of ERBB2 in human myocardium.

Asunto(s)

Metilación de ADN , Regulación de la Expresión Génica , Miocardio/metabolismo , Receptor ErbB-2/genética , Sitios de Unión , Línea Celular , Supervivencia Celular/genética , Islas de CpG , Sitios Genéticos , Humanos , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , Receptor ErbB-2/metabolismo , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional

RESUMEN

BACKGROUND: Tissue engineering enables the generation of functional human cardiac tissue with cells derived in vitro in combination with biocompatible materials. Human-induced pluripotent stem cell-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electric pacing on the maturation of human-induced pluripotent stem cell-derived cardiac tissues. METHODS: Cardiomyocytes derived from human-induced pluripotent stem cells were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electric pacing conditions. RESULTS: The engineered human myocardium exhibits Frank-Starling-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm2 vs 0.055±0.009 mN/mm2 for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 (Sarco/Endoplasmic Reticulum Calcium ATPase 2) expression, which correlated with a less negative force-frequency relationship. When electric pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm2), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 (Ryanodine Receptor 2) and SERCA2 further increased by combined static stress and electric stimulation. CONCLUSIONS: These studies demonstrate that electric pacing and mechanical stimulation promote maturation of the structural, mechanical, and force generation properties of human-induced pluripotent stem cell-derived cardiac tissues.

Asunto(s)

Células Madre Pluripotentes Inducidas/fisiología , Miocardio/metabolismo , Miocitos Cardíacos/fisiología , Ingeniería de Tejidos/métodos , Animales , Humanos , Estrés Mecánico

RESUMEN

BACKGROUND: The postnatal development of myofibrillar mechanics, a major determinant of heart function, is unknown in pediatric patients with tetralogy of Fallot and related structural heart defects. We therefore determined the mechanical properties of myofibrils isolated from right ventricular tissue samples from such patients in relation to the developmental changes of the isoforms expression pattern of key sarcomere proteins involved in the contractile process. METHODS AND RESULTS: Tissue samples from the infundibulum obtained during surgery from 25 patients (age range 15 days to 11 years, median 7 months) were split into half for mechanical investigations and expression analysis of titin, myosin heavy and light chain 1, troponin-T, and troponin-I. Of these proteins, fetal isoforms of only myosin light chain 1 (ALC-1) and troponin-I (ssTnI) were highly expressed in neonates, amounting to, respectively, 40% and 80%, while the other proteins had switched to the adult isoforms before or around birth. ALC-1 and ssTnI expression subsequently declined monoexponentially with a halftime of 4.3 and 5.8 months, respectively. Coincident with the expression of ssTnI, Ca(2+) sensitivity of contraction was high in neonates and subsequently declined in parallel with the decline in ssTnI expression. Passive tension positively correlated with Ca(2+) sensitivity but not with titin expression. Contraction kinetics, maximal Ca(2+)-activated force, and the fast phase of the biphasic relaxation positively correlated with the expression of ALC-1. CONCLUSIONS: The developmental changes in myofibrillar biomechanics can be ascribed to fetal-to-adult isoform transition of key sarcomeric proteins, which evolves regardless of the specific congenital cardiac malformations in our pediatric patients.

Asunto(s)

Cardiopatías Congénitas/fisiopatología , Miofibrillas/fisiología , Fenómenos Biomecánicos/fisiología , Niño , Preescolar , Conectina/metabolismo , Corazón/crecimiento & desarrollo , Humanos , Lactante , Recién Nacido , Proteínas Musculares/fisiología , Contracción Miocárdica/fisiología , Cadenas Pesadas de Miosina/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Sarcómeros/fisiología , Troponina I/metabolismo , Troponina T/metabolismo

RESUMEN

Morphological and histochemical analysis of the heart is fundamental for the understanding of cardiac physiology and pathology. The accurate detection of different myocardial cell populations, as well as the high-resolution imaging of protein expression and distribution, within the diverse intracellular compartments, is essential for basic research on disease mechanisms and for the translatability of the results to human pathophysiology. While enormous progress has been made on the imaging hardware and methods and on biotechnological tools [e.g., use of green fluorescent protein (GFP), viral-mediated gene transduction] to investigate heart cell structure and function, most of the protocols to prepare heart tissue samples for analysis have remained almost identical for decades. We here provide a detailed description of a novel protocol of heart processing, tailored to the simultaneous detection of tissue morphology, immunofluorescence markers and native emission of fluorescent proteins (i.e., GFP). We compared a variety of procedures of fixation, antigen unmasking and tissue permeabilization, to identify the best combination for preservation of myocardial morphology and native GFP fluorescence, while simultaneously allowing detection of antibody staining toward sarcomeric, membrane, cytosolic and nuclear markers. Furthermore, with minimal variations, we implemented such protocol for the study of human heart samples, including those already fixed and stored with conventional procedures, in tissue archives or bio-banks. In conclusion, a procedure is here presented for the laboratory investigation of the heart, in both rodents and humans, which accrues from the same tissue section information that would normally require the time-consuming and tissue-wasting observation of multiple serial sections.

Asunto(s)

Proteínas Fluorescentes Verdes/análisis , Corazón , Inmunohistoquímica/métodos , Miocardio/metabolismo , Animales , Fluorescencia , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microondas , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas

RESUMEN

Claudin-5 is transcriptionally downregulated resulting in dramatically reduced protein levels in human heart failure. Studies in mice have demonstrated that reduced claudin-5 levels occur prior to cardiac damage and far before reduced whole heart function. Therefore, claudin-5 may be a useful early therapeutic target for human heart failure. However, the cell types in which claudin-5 is localized in human heart and from which claudin-5 is reduced in heart failure is not known. The recent identification of claudin-5's interaction with ephrin-B1 in mouse hearts has also not been investigated in non-failing or failing human hearts. In this study we collected human left ventricular mid-myocardium histological samples from 7 non-failing hearts and 16 end-stage failing hearts. Immunoblots demonstrate severe reductions of claudin-5 protein in 14 of 16 failing hearts compared to non-failing controls. Claudin-5 was observed to localize to cardiomyocytes, endothelial cells, and a subset of fibroblasts in non-failing human heart sections. In isolated cardiomyocytes, the transmembrane claudin-5 protein localized in longitudinal striations in lateral membranes. In failing heart, both cardiomyocyte and endothelial claudin-5 localization was severely reduced, but claudin-5 remained in fibroblasts. Absence of claudin-5 staining also correlated with the reduction of the endothelial cell marker CD31. Ephrin-B1 localization, but not protein levels, was altered in failing hearts supporting that claudin-5 is required for ephrin-B1 localization. These data support that loss of claudin-5 in cardiomyocytes and endothelial cells is prevalent in human heart failure. Investigating claudin-5/ephrin-B1 protein complexes and gene regulation may lead to novel therapies.

Asunto(s)

Claudina-5/metabolismo , Células Endoteliales/metabolismo , Efrina-B1/metabolismo , Fibroblastos/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Adulto , Anciano , Western Blotting , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

BACKGROUND: Ischemic postconditioning (IPostC), has been proposed as a useful approach to reduce infarct size in all species, but its clinical utility remains unclear. OBJECTIVE: To investigate the role played by the protocol used on the efficacy of IPostC in protecting the diseased human myocardium. METHODS: Myocardial atrial samples from patients were subjected to a 90 min ischemia/120 min reoxygenation followed by different IPostC protocols to investigate the role of the time of ischemia (30, 60, 90 and 120 s) and the number of cycles (1, 2, 3 and 4) with 60 and 120 s of total ischemic time. Muscles were also subjected to ischemic preconditioning (IPreC). The release of lactate dehydrogenase (LDH) and the measurement of tetrazolium bromide (MTT) were determined. RESULTS: IPostC increased the LDH and decreased the MTT values from those of control, independently of the duration of the conditioning ischemia. LDH and MTT values also worsened by augmenting the number of IPostC cycles whereas they were significantly improved by IPreC. However, analysis of individual results indicated that in approximately 1/3 of the cases IPostC exhibited some degree of protection especially in the presence of increased ischemic injury. CONCLUSIONS: The present findings show that IPostC of the human myocardium may be influenced by the protocol used and also by the degree of the preceding ischemic injury. IPostC was beneficial in approximately 1/3 of the cases; however in the remaining cases it increased ischemic damage and, therefore, these results raise a word of caution on its broad clinical use.

RESUMEN

OBJECTIVES: The aim of this study was to determine whether altered calreticulin expression and distribution contribute to the pathogenesis of atrial fibrillation (AF) associated with valvular heart disease (VHD). BACKGROUND: AF affects electrophysiological and structural changes that exacerbate AF. Atrial remodeling reportedly underlies AF generation, but the precise mechanism of atrial remodeling in AF remains unclear. METHODS: Right and left atrial specimens were obtained from 68 patients undergoing valve replacement surgery. The patients were divided into sinus rhythm (SR; n=25), paroxysmal AF (PaAF; n=11), and persistent AF (PeAF; AF lasting >6 months; n=32) groups. Calreticulin, integrin-α5, and transforming growth factor-ß1 (TGF-ß1) mRNA and protein expression were measured. We also performed immunoprecipitation for calreticulin with either calcineurin B or integrin-α5. RESULTS: Calreticulin, integrin-α5, and TGF-ß1 mRNA and protein expression were increased in the AF groups, especially in the left atrium in patients with mitral valve disease. Calreticulin interacted with both calcineurin B and integrin-α5. Integrin-α5 expression correlated with TGF-ß1 expression, while calreticulin expression correlated with integrin-α5 and TGF-ß1 expression. Despite similar cardiac function classifications, calreticulin expression was greater in the PeAF group than in the SR group. CONCLUSIONS: Calreticulin, integrin-α5, and TGF-ß1 expression was increased in atrial tissue in patients with AF and was related to AF type, suggesting that calreticulin is involved in the pathogenesis of AF in VHD patients.

Asunto(s)

Fibrilación Atrial/genética , Calreticulina/genética , Regulación de la Expresión Génica , Atrios Cardíacos/metabolismo , Enfermedades de las Válvulas Cardíacas/genética , Integrina alfa5/genética , Factor de Crecimiento Transformador beta1/genética , Adolescente , Adulto , Fibrilación Atrial/etiología , Fibrilación Atrial/metabolismo , Western Blotting , Calreticulina/biosíntesis , Femenino , Estudios de Seguimiento , Atrios Cardíacos/patología , Enfermedades de las Válvulas Cardíacas/complicaciones , Enfermedades de las Válvulas Cardíacas/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Integrina alfa5/biosíntesis , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cardiopatía Reumática/complicaciones , Cardiopatía Reumática/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Adulto Joven

RESUMEN

Nonsarcomeric alpha-actinin (ACTN-1)-positive clusters have been detected in human myocardium structurally jeopardized by dilated cardiomyopathy, hypertrophy due to aortic stenosis, or chronic hibernation, but have never been detected in normal tissue. To systematically investigate these clusters, immunohistochemistry, electron microscopy, Northern blot and Western blot were performed in human myocardium, isolated rat cardiomyocytes and rabbit smooth muscle cells. ACTN-1-positive clusters were localized in the perinuclear area of cardiomyocytes surrounded by rough endoplasmic reticulum. Quantification of structures containing ACTN-1 showed that it was present in up to 10% of all myocytes in 60% of aortic stenosis patients with severely reduced ejection fraction and in 70% of patients with dilated cardiomyopathy, exclusively in myocytes from hearts with structural degeneration and reduced function. Ultrastructurally, clusters of medium electron density corresponding to the confocal microscopic accumulations were observed in the same tissue samples. The messenger RNA of ACTN-1 was unchanged compared with controls, but a Western blot revealed that the protein was significantly elevated in failing hearts. Because membranes of the endoplasmic reticulum surround the clusters, it was concluded that in the presence of undisturbed transcription, a post-translational malfunction of ACTN-1 glycosylation might lead to storage of this protein. Autophagic and ischemic cell death were observed, but a possible toxic effect of this storage product was excluded because markers of cell death rarely colocalized with ACTN-1. The occurrence of ACTN-1-positive clusters, however, appears to be a useful marker for structural degeneration in failing myocardium.