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BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetic heart disease associated with life-threatening ventricular arrhythmias. Diagnosis of ARVC is based on the 2010 Task Force Criteria (TFC), application of which often requires clinical expertise at specialized centers. OBJECTIVE: To develop and validate an electrocardiogram (ECG) deep-learning (DL) tool for ARVC diagnosis. METHODS: ECGs of patients referred for ARVC evaluation were used to develop (n=551, 80.1%) and test (n=137, 19.9%) an ECG-DL model for prediction of TFC-defined ARVC diagnosis. The ARVC ECG-DL model was externally validated in a cohort of patients with pathogenic or likely pathogenic (P/LP) ARVC gene variants identified through the Geisinger MyCode Community Health Initiative (N=167). RESULTS: Of 688 patients evaluated at JHH (57.3% male, mean age 40.2 years), 329 (47.8%) were diagnosed with ARVC. While ARVC diagnosis made by referring cardiologist ECG interpretation was unreliable (c-statistic 0.53 [CI: 0.52, 0.53]), ECG-DL discrimination in the hold-out testing cohort was excellent (0.87 [0.86, 0.89]) and compared favorably to that of ECG interpretation by an ARVC expert (0.85 [0.84, 0.86]). In the Geisinger cohort, prevalence of ARVC was lower (n=17, 10.2%), but ECG-DL based identification of ARVC phenotype remained reliable (0.80 [0.77, 0.83]). Discrimination was further increased when ECG-DL predictions were combined with non-ECG-derived TFC in the JHH testing (c-statistic 0.940 [95%CI: 0.933; 0.948]) and Geisinger validation (0.897 [95%CI: 0.883; 0.912]) cohorts. CONCLUSION: ECG-DL augments diagnosis of ARVC to the level of an ARVC expert and can differentiate true ARVC diagnosis from phenotype-mimics and at-risk family members/genotype-positive individuals.
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BACKGROUND AND AIMS: Pathogenic desmoplakin (DSP) gene variants are associated with the development of a distinct form of arrhythmogenic cardiomyopathy known as DSP cardiomyopathy. Patients harbouring these variants are at high risk for sustained ventricular arrhythmia (VA), but existing tools for individualized arrhythmic risk assessment have proven unreliable in this population. METHODS: Patients from the multi-national DSP-ERADOS (Desmoplakin SPecific Effort for a RAre Disease Outcome Study) Network patient registry who had pathogenic or likely pathogenic DSP variants and no sustained VA prior to enrolment were followed longitudinally for the development of first sustained VA event. Clinically guided, step-wise Cox regression analysis was used to develop a novel clinical tool predicting the development of incident VA. Model performance was assessed by c-statistic in both the model development cohort (n = 385) and in an external validation cohort (n = 86). RESULTS: In total, 471 DSP patients [mean age 37.8 years, 65.6% women, 38.6% probands, 26% with left ventricular ejection fraction (LVEF) < 50%] were followed for a median of 4.0 (interquartile range: 1.6-7.3) years; 71 experienced first sustained VA events {2.6% [95% confidence interval (CI): 2.0, 3.5] events/year}. Within the development cohort, five readily available clinical parameters were identified as independent predictors of VA and included in a novel DSP risk score: female sex [hazard ratio (HR) 1.9 (95% CI: 1.1-3.4)], history of non-sustained ventricular tachycardia [HR 1.7 (95% CI: 1.1-2.8)], natural logarithm of 24-h premature ventricular contraction burden [HR 1.3 (95% CI: 1.1-1.4)], LVEF < 50% [HR 1.5 (95% CI: .95-2.5)], and presence of moderate to severe right ventricular systolic dysfunction [HR 6.0 (95% CI: 2.9-12.5)]. The model demonstrated good risk discrimination within both the development [c-statistic .782 (95% CI: .77-.80)] and external validation [c-statistic .791 (95% CI: .75-.83)] cohorts. The negative predictive value for DSP patients in the external validation cohort deemed to be at low risk for VA (<5% at 5 years; n = 26) was 100%. CONCLUSIONS: The DSP risk score is a novel model that leverages readily available clinical parameters to provide individualized VA risk assessment for DSP patients. This tool may help guide decision-making for primary prevention implantable cardioverter-defibrillator placement in this high-risk population and supports a gene-first risk stratification approach.
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Desmoplaquinas , Humanos , Desmoplaquinas/genética , Femenino , Masculino , Medición de Riesgo/métodos , Adulto , Persona de Mediana Edad , Arritmias Cardíacas/genética , Heterocigoto , Taquicardia Ventricular/genéticaRESUMEN
Heart failure (HF) is a complex trait, influenced by environmental and genetic factors, that affects over 30 million individuals worldwide. Historically, the genetics of HF have been studied in Mendelian forms of disease, where rare genetic variants have been linked to familial cardiomyopathies. More recently, genome-wide association studies (GWAS) have successfully identified common genetic variants associated with risk of HF. However, the relative importance of genetic variants across the allele-frequency spectrum remains incompletely characterized. Here, we report the results of common- and rare-variant association studies of all-cause heart failure, applying recently developed methods to quantify the heritability of HF attributable to different classes of genetic variation. We combine GWAS data across multiple populations including 207,346 individuals with HF and 2,151,210 without, identifying 176 risk loci at genome-wide significance (p < 5×10-8). Signals at newly identified common-variant loci include coding variants in Mendelian cardiomyopathy genes (MYBPC3, BAG3), as well as regulators of lipoprotein (LPL) and glucose metabolism (GIPR, GLP1R), and are enriched in cardiac, muscle, nerve, and vascular tissues, as well as myocyte and adipocyte cell types. Gene burden studies across three biobanks (PMBB, UKB, AOU) including 27,208 individuals with HF and 349,126 without uncover exome-wide significant (p < 3.15×10-6) associations for HF and rare predicted loss-of-function (pLoF) variants in TTN, MYBPC3, FLNC, and BAG3. Total burden heritability of rare coding variants (2.2%, 95% CI 0.99-3.5%) is highly concentrated in a small set of Mendelian cardiomyopathy genes, and is lower than heritability attributable to common variants (4.3%, 95% CI 3.9-4.7%) which is more diffusely spread throughout the genome. Finally, we demonstrate that common-variant background, in the form of a polygenic risk score (PRS), significantly modifies the risk of HF among carriers of pathogenic truncating variants in the Mendelian cardiomyopathy gene TTN. These findings suggest a significant polygenic component to HF exists that is not captured by current clinical genetic testing.
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BACKGROUND: The FLNC gene has recently garnered attention as a likely cause of arrhythmogenic cardiomyopathy, which is considered an actionable genetic condition. However, the association with disease in an unselected clinical population is unknown. We hypothesized that individuals with loss-of-function variants in FLNC (FLNCLOF) would have increased odds for arrhythmogenic cardiomyopathy-associated phenotypes versus variant-negative controls in the Geisinger MyCode cohort. METHODS: We identified rare, putative FLNCLOF among 171 948 individuals with exome sequencing linked to health records. Associations with arrhythmogenic cardiomyopathy phenotypes from available diagnoses and cardiac evaluations were investigated. RESULTS: Sixty individuals (0.03%; median age 58 years [47-70 interquartile range], 43% male) harbored 27 unique FLNCLOF. These individuals had significantly increased odds ratios for dilated cardiomyopathy (odds ratio, 4.9 [95% CI, 2.6-7.6]; P<0.001), supraventricular tachycardia (odds ratio, 3.2 [95% CI, 1.1-5.6]; P=0.048), and left-dominant arrhythmogenic cardiomyopathy (odds ratio, 4.2 [95% CI, 1.4-7.9]; P=0.03). Echocardiography revealed reduced left ventricular ejection fraction (52±13% versus 57±9%; P=0.001) associated with FLNCLOF. Overall, at least 9% of FLNCLOF patients demonstrated evidence of penetrant disease. CONCLUSIONS: FLNCLOF variants are associated with increased odds of ventricular arrhythmia and dysfunction in an unselected clinical population. These findings support genomic screening of FLNC for actionable secondary findings.
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Cardiomiopatía Dilatada , Filaminas , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/genética , Cardiomiopatía Dilatada/complicaciones , Cardiomiopatía Dilatada/genética , Exoma , Femenino , Filaminas/genética , Humanos , Masculino , Fenotipo , Volumen Sistólico , Función Ventricular Izquierda , Secuenciación del ExomaRESUMEN
PURPOSE: Right ventricular (RV) function is increasingly recognized for its prognostic value in many disease states. As with the left ventricle (LV), strain-based measurements may have better prognostic value than typical chamber volumes or ejection fraction. Complete functional characterization of the RV requires high-resolution, 3D displacement tracking methods, which have been prohibitively challenging to implement. Zonal excitation during Displacement ENcoding with Stimulated Echoes (DENSE) magnetic resonance imaging (MRI) has helped reduce scan time for 2D LV strain quantification. We hypothesized that zonal excitation could alternatively be used to reproducibly acquire higher resolution, 3D-encoded DENSE images for quantification of bi-ventricular strain within a single breath-hold. METHODS: We modified sequence parameters for a 3D zonal excitation DENSE sequence to achieve in-plane resolution < 2 mm and acquired two sets of images in eight healthy adult male volunteers with median (IQR) age 32.5 (32.0-33.8) years. We assessed the inter-test reproducibility of this technique, and compared computed strains and torsion with previously published data. RESULTS: Data for one subject was excluded based on image artifacts. Reproducibility for LV (CoV: 6.1-9.0%) and RV normal strains (CoV: 6.3-8.2%) and LV torsion (CoV = 7.1%) were all very good. Reproducibility of RV torsion was lower (CoV = 16.7%), but still within acceptable limits. Computed global strains and torsion were within reasonable agreement with published data, but further studies in larger cohorts are needed to confirm. CONCLUSION: Reproducible acquisition of 3D-encoded biventricular myocardial strain data in a breath-hold is feasible using DENSE with zonal excitation.
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Interpretación de Imagen Asistida por Computador , Imagen por Resonancia Cinemagnética , Adulto , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética , Masculino , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Función Ventricular IzquierdaRESUMEN
BACKGROUND: Genomic screening holds great promise for presymptomatic identification of hidden disease, and prevention of dramatic events, including sudden cardiac death associated with arrhythmogenic cardiomyopathy (ACM). Herein, we present findings from clinical follow-up of carriers of ACM-associated pathogenic/likely pathogenic desmosome variants ascertained through genomic screening. METHODS: Of 64 548 eligible participants in Geisinger MyCode Genomic Screening and Counseling program (2015-present), 92 individuals (0.14%) identified with pathogenic/likely pathogenic desmosome variants by clinical laboratory testing were referred for evaluation. We reviewed preresult medical history, patient-reported family history, and diagnostic testing results to assess both arrhythmogenic right ventricular cardiomyopathy and left-dominant ACM. RESULTS: One carrier had a prior diagnosis of dilated cardiomyopathy with arrhythmia; no other related diagnoses or diagnostic family history criteria were reported. Fifty-nine carriers (64%) had diagnostic testing in follow-up. Excluding the variant, 21/59 carriers satisfied at least one arrhythmogenic right ventricular cardiomyopathy task force criterion, 11 (52%) of whom harbored DSP variants, but only 5 exhibited multiple criteria. Six (10%) carriers demonstrated evidence of left-dominant ACM, including high rates of atypical late gadolinium enhancement by magnetic resonance imaging and nonsustained ventricular tachycardia. Two individuals received new cardiomyopathy diagnoses and received defibrillators for primary prevention. CONCLUSIONS: Genomic screening for pathogenic/likely pathogenic variants in desmosome genes can uncover both left- and right-dominant ACM. Findings of overt cardiomyopathy were limited but were most common in DSP-variant carriers and notably absent in PKP2-variant carriers. Consideration of the pathogenic/likely pathogenic variant as a major criterion for diagnosis is inappropriate in the setting of genomic screening.
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Displasia Ventricular Derecha Arritmogénica/diagnóstico , Desmosomas/genética , Variación Genética , Adulto , Anciano , Displasia Ventricular Derecha Arritmogénica/genética , Displasia Ventricular Derecha Arritmogénica/patología , Desmocolinas/genética , Desmogleína 2/genética , Ecocardiografía , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Placofilinas/genéticaRESUMEN
BACKGROUND: Pressure overload left ventricular (LV) hypertrophy is characterized by increased cardiomyocyte width and ventricle wall thickness, however the regional variation of this remodeling is unclear. Cardiovascular magnetic resonance (CMR) diffusion tensor imaging (DTI) may provide a non-invasive, comprehensive, and geometrically accurate method to detect regional differences in structural remodeling in hypertrophy. We hypothesized that DTI parameters, such as fractional and planar anisotropy, would reflect myocyte remodeling due to pressure overload in a regionally-dependent manner. METHODS: We investigated the regional distributions of myocyte remodeling in rats with or without transverse aortic constriction (TAC) via direct measurement of myocyte dimensions with confocal imaging of thick tissue sections, and correlated myocyte cross-sectional area and other geometric features with parameters of diffusivity from ex-vivo DTI in the same regions of the same hearts. RESULTS: We observed regional differences in several parameters from DTI between TAC hearts and SHAM controls. Consistent with previous studies, helix angles from DTI correlated strongly with those measured directly from histological sections (p < 0.001, R2 = 0.71). There was a transmural gradient in myocyte cross-sectional area in SHAM hearts that was diminished in the TAC group. We also found several regions of significantly altered DTI parameters in TAC LV compared to SHAM, especially in myocyte sheet angle dispersion and planar anisotropy. Among others, these parameters correlated significantly with directly measured myocyte aspect ratios. CONCLUSIONS: These results show that structural remodeling in pressure overload LV hypertrophy is regionally heterogeneous, especially transmurally, with a greater degree of remodeling in the sub-endocardium compared to the sub-epicardium. Additionally, several parameters derived from DTI correlated significantly with measurements of myocyte geometry from direct measurement in histological sections. We suggest that DTI may provide a non-invasive, comprehensive method to detect regional structural myocyte LV remodeling during disease.
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Tamaño de la Célula , Imagen de Difusión Tensora , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Miocitos Cardíacos/patología , Función Ventricular Izquierda , Presión Ventricular , Remodelación Ventricular , Animales , Modelos Animales de Enfermedad , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Valor Predictivo de las Pruebas , Ratas Sprague-DawleyRESUMEN
PURPOSE: Structure-guided total variation is a recently introduced prior that allows reconstruction of images using knowledge of the location and orientation of edges in a reference image. In this work, we demonstrate the advantages of a variant of structure-guided total variation known as directional total variation (DTV), over traditional total variation (TV), in the context of compressed-sensing reconstruction and super-resolution. METHODS: We compared TV and DTV in retrospectively undersampled ex vivo diffusion tensor imaging and diffusion spectrum imaging data from healthy, sham, and hypertrophic rat hearts. RESULTS: In compressed sensing at an undersampling factor of 8, the RMS error of mean diffusivity and fractional anisotropy relative to the fully sampled ground truth were 44% and 20% lower in DTV compared with TV. In super-resolution, these values were 29% and 14%, respectively. Similarly, we observed improvements in helix angle, transverse angle, sheetlet elevation, and sheetlet azimuth. The RMS error of the diffusion kurtosis in the undersampled data relative to the ground truth was uniformly lower (22% on average) with DTV compared to TV. CONCLUSION: Acquiring one fully sampled non-diffusion-weighted image and 10 diffusion-weighted images at 8× undersampling would result in an 80% net reduction in data needed. We demonstrate efficacy of the DTV algorithm over TV in reducing data sampling requirements, which can be translated into higher apparent resolution and potentially shorter scan times. This method would be equally applicable in diffusion MRI applications outside the heart.
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Imagen de Difusión por Resonancia Magnética , Imagen de Difusión Tensora , Algoritmos , Animales , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Ratas , Estudios RetrospectivosRESUMEN
BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with variants in desmosome genes. Secondary findings of pathogenic/likely pathogenic variants, primarily loss-of-function (LOF) variants, are recommended for clinical reporting; however, their prevalence and associated phenotype in a general clinical population are not fully characterized. METHODS: From whole-exome sequencing of 61 019 individuals in the DiscovEHR cohort, we screened for putative loss-of-function variants in PKP2, DSC2, DSG2, and DSP. We evaluated measures from prior clinical ECG and echocardiograms, manually over-read to evaluate ARVC diagnostic criteria, and performed a PheWAS (phenome-wide association study). Finally, we estimated expected penetrance using Bayesian inference. RESULTS: One hundred forty individuals (0.23%; 59±18 years old at last encounter; 33% male) had an ARVC variant (G+). None had an existing diagnosis of ARVC in the electronic health record, nor significant differences in prior ECG or echocardiogram findings compared with matched controls without variants. Several G+ individuals satisfied major repolarization (n=4) and ventricular function (n=5) criteria, but this prevalence matched controls. PheWAS showed no significant associations of other heart disease diagnoses. Combining our best genetic and disease prevalence estimates yields an estimated penetrance of 6.0%. CONCLUSIONS: The prevalence of ARVC loss-of-function variants is ≈1:435 in a general clinical population of predominantly European descent, but with limited electronic health record-based evidence of phenotypic association in our population, consistent with a low penetrance estimate. Prospective deep phenotyping and longitudinal follow-up of a large sequenced cohort is needed to determine the true clinical relevance of an incidentally identified ARVC loss-of-function variant.
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Displasia Ventricular Derecha Arritmogénica/genética , Registros Electrónicos de Salud/estadística & datos numéricos , Adulto , Anciano , Desmocolinas/genética , Desmogleína 2/genética , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Fenotipo , Placofilinas/genética , Estudios ProspectivosRESUMEN
The cardiac system compensates for variations in physiological and pathophysiological conditions through a dynamic remodeling at the organ, tissue, and intracellular levels in order to maintain function. However, on longer time scales following the onset of ventricular pressure overload, such remodeling may begin to inhibit physiological function and ultimately lead to heart failure. This progression from compensatory to decompensatory behavior is poorly understood, in particular owing to the absence of a unified perspective of the concomitantly remodeling subsystems. To address this issue, the present study investigates the evolution of compensatory mechanisms, in response to overload, by integrating diffusion-tensor MRI, echocardiography, and intracellular and hemodynamic measurements within consistent computational simulations of aortic-banded rat hearts. This approach allows a comparison of the relative leverage of different cardiac properties (geometry, passive mechanical stiffness, fiber configuration, diastolic and peak calcium concentrations, calcium-binding affinity, and aortic impedance) to affect cardiac contraction. Measurements indicate that, following aortic banding, an ejection fraction (EF) of 75% was maintained, relative to control rats, despite significant remodeling of the left-ventricular wall thickness (increasing by ~90% over 4 weeks). Applying our framework, we identified the left-ventricular wall thickness (concentric hypertrophy) and the intracellular calcium dynamics as playing the dominant roles in preserving EF acutely, whereas the significance of hypertrophy decreased subsequently. This trend suggests an increasing reliance on intracellular mechanisms (average increase ~50%), rather than on anatomical features (average decrease ~60%), to achieve compensation of pump function in the early phase of heart failure.
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PURPOSE: The diffusion tensor model assumes Gaussian diffusion and is widely applied in cardiac diffusion MRI. However, diffusion in biological tissue deviates from a Gaussian profile as a result of hindrance and restriction from cell and tissue microstructure, and may be quantified better by non-Gaussian modeling. The aim of this study was to investigate non-Gaussian diffusion in healthy and hypertrophic hearts. METHODS: Thirteen rat hearts (five healthy, four sham, four hypertrophic) were imaged ex vivo. Diffusion-weighted images were acquired at b-values up to 10,000 s/mm2 . Models of diffusion were fit to the data and ranked based on the Akaike information criterion. RESULTS: The diffusion tensor was ranked best at b-values up to 2000 s/mm2 but reflected the signal poorly in the high b-value regime, in which the best model was a non-Gaussian "beta distribution" model. Although there was considerable overlap in apparent diffusivities between the healthy, sham, and hypertrophic hearts, diffusion kurtosis and skewness in the hypertrophic hearts were more than 20% higher in the sheetlet and sheetlet-normal directions. CONCLUSION: Non-Gaussian diffusion models have a higher sensitivity for the detection of hypertrophy compared with the Gaussian model. In particular, diffusion kurtosis may serve as a useful biomarker for characterization of disease and remodeling in the heart. Magn Reson Med 78:1174-1186, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
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Imagen de Difusión por Resonancia Magnética/métodos , Corazón/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Animales , Cardiomegalia/diagnóstico por imagen , Masculino , Distribución Normal , Ratas , Ratas Sprague-DawleyRESUMEN
Although a truly complete understanding of whole heart activation, contraction, and deformation is well beyond our current reach, a significant amount of effort has been devoted to discovering and understanding the mechanisms by which myocardial structure determines cardiac function to better treat patients with cardiac disease. Several experimental studies have shown that transmural fiber strain is relatively uniform in both diastole and systole, in contrast to predictions from traditional mechanical theory. Similarly, mathematical models have largely predicted uniform fiber stress across the wall. The development of this uniform pattern of fiber stress and strain during filling and ejection is due to heterogeneous transmural distributions of several myocardial structures. This review summarizes these transmural gradients, their contributions to fiber mechanics, and the potential functional effects of their remodeling during pressure overload hypertrophy.
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Miocardio/citología , Presión/efectos adversos , Estrés Mecánico , Animales , Corazón/fisiología , HumanosRESUMEN
Sarcomeres are the basic contractile units of cardiac myocytes. Recent studies demonstrated remodeling of sarcomeric proteins in several diseases, including genetic defects and heart failure. Here we investigated remodeling of sarcomeric α-actinin in two models of heart failure, synchronous (SHF) and dyssynchronous heart failure (DHF), as well as a model of cardiac resynchronization therapy (CRT). We applied three-dimensional confocal microscopy and quantitative methods of image analysis to study isolated cells from our animal models. 3D Fourier analysis revealed a decrease of the spatial regularity of the α-actinin distribution in both SHF and DHF versus control cells. The spatial regularity of α-actinin in DHF cells was reduced when compared with SHF cells. The spatial regularity of α-actinin was partially restored after CRT. We found longitudinal depositions of α-actinin in SHF, DHF and CRT cells. These depositions spanned adjacent Z-disks and exhibited a lower density of α-actinin than in the Z-disk. Differences in the occurrence of depositions between the SHF, CRT and DHF models versus control were significant. Also, CRT cells exhibited a higher occurrence of depositions versus SHF, but not DHF cells. Other sarcomeric proteins did not accumulate in the depositions to the same extent as α-actinin. We did not find differences in the expression of α-actinin protein and its encoding gene in our animal models. In summary, our studies indicate that HF is associated with two different types of remodeling of α-actinin and only one of those was reversed after CRT. We suggest that these results can guide us to an understanding of remodeling of structures and function associated with sarcomeres.
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Actinina/química , Terapia de Resincronización Cardíaca , Citoesqueleto/ultraestructura , Insuficiencia Cardíaca/terapia , Ventrículos Cardíacos/ultraestructura , Miocitos Cardíacos/ultraestructura , Actinina/genética , Actinina/metabolismo , Animales , Citoesqueleto/metabolismo , Citoesqueleto/patología , Perros , Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Masculino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Sarcómeros/metabolismo , Sarcómeros/patología , Sarcómeros/ultraestructura , Remodelación VentricularRESUMEN
Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution. We applied this methodology to normal rabbit ventricular tissue and tissue from hearts with myocardial infarction. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83±0.42% (mean ± standard deviation) in normal tissue up to 6.51±0.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.20±9.89% in normal to 73.48±8.02% adjacent to the infarct. Numerical field calculations on 3-D reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.264±0.082 S/m with an anisotropy ratio of 2.095±1.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.400±0.051 S/m, but the anisotropy ratio decreased to 1.295±0.09. Our study indicates an increased density of gap junctions proximal to both fibroblasts and myocytes in infarcted versus normal tissue, supporting previous hypotheses of electrical coupling of fibroblasts and myocytes in infarcted hearts. We suggest that the presented methodology provides an important contribution to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease-associated remodeling.
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Corazón/fisiología , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Modelos Cardiovasculares , Miocardio/química , Miocardio/citología , Animales , Conexinas/química , Conductividad Eléctrica , Fenómenos Electrofisiológicos , Fibroblastos/citología , Colorantes Fluorescentes/química , Uniones Comunicantes/química , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/citología , ConejosRESUMEN
Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.