Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2±0.3% and 98.0±0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2±0.3% and 94.0±2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.     

Matrix metalloproteinases as candidate biomarkers in adults with congenital heart disease

Context Matrix metalloproteinases (MMPs) are associated with diastolic dysfunction and heart failure in acquired heart disease.

Objective To investigate the role of MMPs as novel biomarkers in clinically stable adults with congenital heart disease.

Methods We measured serum MMP-2, -3, -9 and tissue inhibitor of matrix metalloproteinase-1 in 425 patients and analysed the association with cardiac function and exercise capacity.

Results MMP-2 was significantly associated with exercise capacity, ventilatory efficiency and left ventricular deceleration time, independently of age, sex, body surface area and NT-proBNP.

Conclusion MMP-2 may provide new information in the clinical evaluation of adults with congenital heart disease.     

cTnT1, a cardiac troponin T isoform, decreases myofilament tension and affects the left ventricular pressure waveform

Four isoforms of cardiac troponin T (cTnT), a protein essential for calcium-dependent myocardial force development, are expressed in the human; they differ in charge and length. Their expression is regulated developmentally and is affected by disease states. Human cTnT (hcTnT) isoform effects have been examined in reconstituted myofilaments. In this study, we evaluated the modulatory effects of overexpressing one cTnT isoform on in vitro and in vivo myocardial function. A hcTnT isoform, hcTnT(1), expressed during development and in heart disease but not in the normal adult heart, was expressed in transgenic (TG) mice (1-30% of total cTnT). Maximal active tension measured in skinned myocardium decreased as a function of relative hcTnT(1) expression. The pCa at half-maximal force development, Hill coefficient, and rate of redevelopment of force did not change significantly with hcTnT(1) expression. In vivo maximum rates of rise and fall of left ventricular pressure decreased, and the half-time of isovolumic relaxation increased, with hcTnT(1) expression. Substituting total cTnT charge for hcTnT(1) expression resulted in similar conclusions. Morphometric analysis and electron microscopy revealed no differences between wild-type (non-TG) and TG myocardium. No differences in isoform expression of tropomyosin, myosin heavy chain, essential and regulatory myosin light chains (MLC), TnI, or in posttranslational modifications of mouse cTnT, cTnI, or regulatory MLC were observed. These results support the hypothesis that cTnT isoform amino-terminal differences affect myofilament function and suggest that hcTnT(1) expression levels present during human development and in human heart disease can affect in vivo ventricular function.     

人类单纯性先天性心脏病中TBX5基因的突变及表达研究

本文首次较为完整地报道了藏汉通婚子代群体的14项肤纹参数（其中藏父汉思及汉父藏母各100 例）,并将这些肤纹参数分别与其藏汉父母样本的有关肤纹参数进行比较,再与1000例藏族及1040例 汉族两个大样本的有关肤纹参数进行比较。结果表明：藏汉后代的肤纹特征介于藏族和汉族之间,提示 肤纹参数的多因子遗传本质。     

Effect of cardioplegia on nitrogen and energy metabolism of the human heart

The interrelation between the energy and nitrogenous metabolism of the myocardium during cardioplegia has been studied in patients with congenital valvular heart disease (tetralogy of Fallot--12 patients, ventricular septal defect--5 patients). Whole body hypothermia with repeated heart reperfusion with cold cardioplegic blood perfusate was used for the protection of the myocardium. However, ATP level of the myocardium of some patients decreased by 20% and more of the baseline. This loss was accompanied by a reduction in glutamate and aspartate levels and a rise in ammonium and alanine levels in the myocardium (by 17.7 +/- 3.8; 17.6 +/- 5.9; 61.4 +/- 12.5 and 92.4 +/- 26.3% of the baseline, respectively).     

Developmental changes in dopamine modulation of the heart in the isopod crustacean Ligia exotica: reversal of chronotropic effect

Developmental changes in dopamine modulation of the heart were examined in the isopod crustacean Ligia exotica. The Ligia cardiac pacemaker is transferred from the myocardium to the cardiac ganglion during juvenile development and the heartbeat changes from myogenic to neurogenic. In the myogenic heart of early juveniles, dopamine affected the myocardium and caused a decrease in the frequency and an increase in the duration of the myocardial action potential, resulting in negative chronotropic (decrease in beat frequency) and positive inotropic (increase in contractile force) effects on the heart. Contrastingly, in the heart of immature adults just after juvenile development, dopamine caused effects of adult type, positive chronotropic and positive inotropic effects on the heart affecting the cardiac ganglion and myocardium. During the middle and late juvenile stages, dopamine caused individually a negative or a positive chronotropic effect on the heart. These results suggest that the chronotropic effect of dopamine on the Ligia heart is reversed from negative to positive in association with the cardiac pacemaker transfer from the myocardium to the cardiac ganglion during juvenile development.     

The anterior heart-forming field: voyage to the arterial pole of the heart

Studies of vertebrate heart development have identified key genes and signalling molecules involved in the formation of a myocardial tube from paired heart-forming fields in splanchnic mesoderm. The posterior region of the paired heart-forming fields subsequently contributes myocardial precursor cells to the inflow region or venous pole of the heart. Recently, a population of myocardial precursor cells in chick and mouse embryos has been identified in pharyngeal mesoderm anterior to the early heart tube. This anterior heart-forming field gives rise to myocardium of the outflow region or arterial pole of the heart. The amniote heart is therefore derived from two myocardial precursor cell populations, which appear to be regulated by distinct genetic programmes. Discovery of the anterior heart-forming field has important implications for the interpretation of cardiac defects in mouse mutants and for the study of human congenital heart disease.     

Functional heterogeneity of the atrial myocardium in patients with congenital and acquired heart defects]

In experiments on atrium trabeculae the heterogeneity of myocardium contractile activity in patients operated on for inborn or acquired heart defects was studied. Contractile activity was assessed in isometric regime of muscle drugs work. The degree of functional heterogeneity was reported to differ in myocardium biopsies from patients with inborn or acquired heart defects. The difference was expressed in susceptibility to stimulating action or electrical impulses and in the degree of the change of myocardium contractile activity. The study of human myocardium functional heterogeneity is likely to present a new approach to increase efficacy of the work of pathologically changed heart muscle.     

Arginyltransferase regulates alpha cardiac actin function, myofibril formation and contractility during heart development

Post-translational arginylation mediated by arginyltransferase (Ate1) is essential for cardiovascular development and angiogenesis in mammals and directly affects myocardium structure in the developing heart. We recently showed that arginylation exerts a number of intracellular effects by modifying proteins involved in the functioning of the actin cytoskeleton and in cell motility. Here, we investigated the role of arginylation in the development and function of cardiac myocytes and their actin-containing structures during embryogenesis. Biochemical and mass spectrometry analyses showed that alpha cardiac actin undergoes arginylation at four sites during development. Ultrastructural analysis of the myofibrils in wild-type and Ate1 knockout mouse hearts showed that the absence of arginylation results in defects in myofibril structure that delay their development and affect the continuity of myofibrils throughout the heart, predicting defects in cardiac contractility. Comparison of cardiac myocytes derived from wild-type and Ate1 knockout mouse embryos revealed that the absence of arginylation results in abnormal beating patterns. Our results demonstrate cell-autonomous cardiac myocyte defects in arginylation knockout mice that lead to severe congenital abnormalities similar to those observed in human disease, and outline a new function of arginylation in the regulation of the actin cytoskeleton in cardiac myocytes.     

Laboratory diagnostics of enterovirus cardiac infections

Diagnostic efficacy of different markers of enterovirus cardiac infection (EVCI) has been evaluated. Testing of clinical samples from patients with myocarditis (n=50), dilatation cardiomyopathy (n=122), ischemic heart disease (n=34) and from healthy donors (n=50) revealed diagnostically significant markers in patients suspected for enterovirus cardiac infection: antienterovirus IgM in the patient's blood serum, the expression of viral proteins by myocardium cells and the presence of genome RNA and replicative intermediate in cardiac cells. The results obtained were used for developing up-to-date scheme of the EVCI diagnosis which included the data from the case history, the preliminary rapid diagnosis and the molecular biological study of the cardiac biopsies.     

Building the mammalian heart from two sources of myocardial cells

Cardiogenesis is an exquisitely sensitive process. Any perturbation in the cells that contribute to the building of the heart leads to cardiac malformations, which frequently result in the death of the embryo. Previously, the myocardium was thought to be derived from a single source of cells. However, the recent identification of a second source of myocardial cells that make an important contribution to the cardiac chambers has modified the classical view of heart formation. It also has an important influence on the interpretation of mutant phenotypes in the mouse, with consequences for the classification and prognosis of human congenital heart defects.     

INDICATIONS FOR ANGIOCARDIOGRAPHY

Angiocardiography is indicated in selected cases of heart disease in which a definite diagnosis cannot be made by ordinary methods or in which there is reasonable expectation that the information so obtained may influence the treatment of the patient. Whenever possible, angiocardiography should be done in conjunction with cardiac catheterization. The main indication for angiocardiography is cyanotic congenital heart disease; primarily those cases in which there is a right-to-left shunt. Angiocardiography is occasionally of value in diagnosis of other types of congenital heart disease and in acquired heart disease.     

A spontaneous model for autoimmune myocarditis using the human MHC molecule HLA-DQ8

Genome-wide analyses have shown that the MHC class II region is the principal locus that confers susceptibility to a number of human autoimmune diseases. Due to the high degree of linkage disequilibrium across the MHC, it has been difficult to dissect the contribution of individual genes to disease susceptibility. As a result, intensive efforts have been made to generate mice transgenic for human class II molecules as models of autoimmune disease. However, in every case, additional manipulations-such as immunization with Ag in adjuvant, expression of immunostimulants on target tissues, or coexpression of TCR transgenes-have been required to induce disease. In this study, we show that expression of the human HLA-DQ8 (DQA1*0301/DQB1*0302) molecule alone in three lines of transgenic nonobese diabetic murine class II-deficient (mII(-/-)) mice results in the spontaneous development of autoimmune myocarditis. The disease shares key features of human myocarditis and was characterized by lymphocytic infiltrates in the myocardium and cardiac myocyte destruction, circulating IgG autoantibodies against cardiac myosin heavy chain, and premature death due to heart failure. We demonstrate that myocarditis could be transferred into healthy HLA-DQ8(+)RAG-1(-/-)mII(-/-) nonobese diabetic recipients with lymphocytes, but not sera. It has been widely thought that autoimmune myocarditis is of infectious etiology, with the immune responses arising secondary to cardiac damage from pathogens. These studies provide direct experimental evidence that spontaneous autoimmune myocarditis can occur in the absence of infection and that expression of HLA-DQ8 confers susceptibility to this organ-specific autoimmune disease.     

Atrial myocardium is the predominant inotropic target of adrenomedullin in the human heart

Adrenomedullin (ADM) is an endogenous peptide with favorable hemodynamic effects in vivo. In this study, we characterized the direct functional effects of ADM in isolated preparations from human atria and ventricles. In electrically stimulated human nonfailing right atrial trabeculae, ADM (0.0001-1 micromol/l) increased force of contraction in a concentration-dependent manner, with a maximal increase by 35 +/- 8% (at 1 micromol/l; P < 0.05). The positive inotropic effect was accompanied by a disproportionate increase in calcium transients assessed by aequorin light emission [by 76 +/- 20%; force/light ratio (DeltaF/DeltaL) 0.58 +/- 0.15]. In contrast, elevation of extracellular calcium (from 2.5 to 3.2 mmol/l) proportionally increased force and aequorin light emission (DeltaF/DeltaL 1.0 +/- 0.1; P < 0.05 vs. ADM). Consistent with a cAMP-dependent mechanism, ADM (1 micromol/l) increased atrial cAMP levels by 90 +/- 12%, and its inotropic effects could be blocked by the protein kinase A (PKA) inhibitor H-89. ADM also exerted positive inotropic effects in failing atrial myocardium and in nonfailing and failing ventricular myocardium. The inotropic response was significantly weaker in ventricular vs. atrial myocardium and in failing vs. nonfailing myocardium. In conclusion, ADM exerts Ca(2+)-dependent positive inotropic effects in human atrial and less-pronounced effects in ventricular myocardium. The inotropic effects are related to increased cAMP levels and stimulation of PKA. In heart failure, the responsiveness to ADM is reduced in atria and ventricles.     

CONGENITAL HEART DISEASE—Comments Regarding Incidence and Natural History

The incidence of congenital heart disease is approximately 1 per cent of all live births. Approximately 60 per cent of patients who die of congenital heart disease do so at less than two years of age. Very few patients with such lesions live beyond 45 years at the very most. In about 70 per cent of patients who are born with cardiac anomalies, the lesions are either of kinds that are already being operated upon successfully or for which operations are now being attempted and often are helpful.