What Is the Optimal Light Source for Optical Mapping Using Voltage- and Calcium-Sensitive Dyes?

Optical mapping is a fluorescence-based physiological method to image spreading of action potential in excitable tissues, such as the heart and central nervous system. Because of the requirements for high speed imaging in low light conditions, highly sensitive high-speed cameras together with an optical system with maximum photon efficiency are required. While the optimization of these two components is relatively straightforward, the choice of the perfect light source is less simple; depending on the other (usually fixed) components, various parameters may acquire different weight in decision-making process. Here we describe the rationale for building an optical mapping setup and consider the relative advantages and disadvantages of three different commonly available light sources: mercury vapor lamp (HBO), xenon lamp (XBO), and light emitting diode (LED). Using the same optical system (fluorescence macroscope) and high-speed camera (Ultima L), we have tested each of the sources for its ability to provide bright and even illumination of the field of view and measured its temporal fluctuations in intensity. Then we used each in the actual optical mapping experiment using isolated, perfused adult mouse heart or chick embryonic heart to determine the actual signal to noise ratio at various acquisition rates. While the LED sources have undergone significant improvements in the recent past, the other alternatives may still surpass them in some parameters, so they may not be the automatic number one choice for every application.

Cardiac Enlargement in the Chick Embryo Induced by Hypothermic Incubation Is Due to a Combination of Hyperplasia and Hypertrophy of Cardiomyocytes.

Hypothermic incubation of chicken eggs leads to smaller embryos with enlarged hearts, originally described as hypertrophic. Over the years, however, accumulated evidence suggested that hyperplasia, rather than hypertrophy, is the predominant mechanism of cardiac growth during the prenatal period. We have thus set to re-evaluate the hypothermia model to precise the exact cellular mechanism behind cardiac enlargement. Fertilized chicken eggs were incubated at either 37.5 °C (normothermia) or 33.5 °C from embryonic day (ED) 13 onward (hypothermia). Sampling was performed at ED17, at which point wet embryo and heart weight were recorded, and the hearts were submitted to histological examination. In agreement with previous results, the hypothermic embryos were 29% smaller and had hearts 18% larger, translating into a 67% increase in the heart to body weight ratio (P < 0.05 for all parameters). The cell size was essentially the same between control and hypothermic hearts in all regions analysed. Likewise, there was no significant relationship between the cell size and heart weight; however, in the hypothermic hearts, there was a trend showing positive correlation between cell sizes in different cardiac regions and heart weight. Proliferation rate, determined on the basis of anti-phosphohistone H3 immunofluorescence, showed an overall increase in the hypothermic group, reaching statistical significance (P = 0.02, t-test) in the right ventricle. The proliferation rate was similar among different regions of the same heart. However, the correlation between the proliferation rate and heart weight was only small (r2 = 0.007 and r2 = 0.234 for the normothermic and hypothermic group, respectively). We thus conclude that hyperplasia is the predominant response mechanism in this volume-overload model; mechanistically, decreased heart rate at lower temperature increases the end-diastolic and stroke volume, minimizing the drop in cardiac output through the Frank- Starling mechanism.

Physiological role of FGF signaling in growth and remodeling of developing cardiovascular system.

Fibroblast growth factor (FGF) signaling plays an important role during embryonic induction and patterning, as well as in modulating proliferative and hypertrophic growth in fetal and adult organs. Hemodynamically induced stretching is a powerful physiological stimulus for embryonic myocyte proliferation. The aim of this study was to assess the effect of FGF2 signaling on growth and vascularization of chick embryonic ventricular wall and its involvement in transmission of mechanical stretch-induced signaling to myocyte growth in vivo. Myocyte proliferation was significantly higher at the 48 h sampling interval in pressure-overloaded hearts. Neither Western blotting, nor immunohistochemistry performed on serial paraffin sections revealed any changes in the amount of myocardial FGF2 at that time point. ELISA showed a significant increase of FGF2 in the serum. Increased amount of FGF2 mRNA in the heart was confirmed by real time PCR. Blocking of FGF signaling by SU5402 led to decreased myocyte proliferation, hemorrhages in the areas of developing vasculature in epicardium and digit tips. FGF2 synthesis is increased in embryonic ventricular cardiomyocytes in response to increased stretch due to pressure overload. Inhibition of FGF signaling impacts also vasculogenesis, pointing to partial functional redundancy in paracrine control of cell proliferation in the developing heart.

Acute temperature effects on function of the chick embryonic heart.

AIM: We analysed the effects of acute temperature change on the beating rate, conduction properties and calcium transients in the chick embryonic heart in vitro and in ovo. METHODS: The effects of temperature change (34, 37 and 40 °C) on calcium dynamics in isolated ED4 chick hearts in vitro were investigated by high-speed calcium optical imaging. For comparison and validation of in vitro measurements, experiments were also performed in ovo using videomicroscopy. Artificial stimulation experiments were performed in vitro and in ovo to uncover conduction limits of heart segments. RESULTS: Decrease in temperature from 37 to 34 °C in vitro led to a 22% drop in heart rate and unchanged amplitude of Ca(2+) transients, compared to a 25% heart rate decrease in ovo. Increase in temperature from 37 to 40 °C in vitro and in ovo led to 20 and 23% increases in heart rate, respectively, and a significant decrease in amplitude of Ca(2+) transients (atrium -35%, ventricle -38%). We observed a wide spectrum of arrhythmias in vitro, of which the most common was atrioventricular (AV) block (57%). There was variability of AV block locations. Pacing experiments in vitro and in ovo suggested that the AV blocks were likely caused by relative tissue hypoxia and not by the tachycardia itself. CONCLUSION: The pacemaker and AV canal are the most temperature-sensitive segments of the embryonic heart. We suggest that the critical point for conduction is the connection of the ventricular trabecular network to the AV canal.

Arrhythmias in the developing heart.

Prevalence of cardiac arrhythmias increases gradually with age; however, specific rhythm disturbances can appear even prior to birth and markedly affect foetal development. Relatively little is known about these disorders, chiefly because of their relative rarity and difficulty in diagnosis. In this review, we cover the most common forms found in human pathology, specifically congenital heart block, pre-excitation, extrasystoles and long QT syndrome. In addition, we cover pertinent literature data from prenatal animal models, providing a glimpse into pathogenesis of arrhythmias and possible strategies for treatment.

Why do we have Purkinje fibers deep in our heart?

Purkinje fibers were the first discovered component of the cardiac conduction system. Originally described in sheep in 1839 as pale subendocardial cells, they were found to be present, although with different morphology, in all mammalian and avian hearts. Here we review differences in their appearance and extent in different species, summarize the current state of knowledge of their function, and provide an update on markers for these cells. Special emphasis is given to popular model species and human anatomy.

Reverse endoventricular artificial obturator in tricuspid valve position. Experimental feasibility research study.

The concept of vena contracta space reduction in tricuspid valve position was tested in an animal model. Feasibility of specific artificial obturator body (REMOT) fixed to the right ventricular apex and interacting with tricuspid valve leaflets was evaluated in three different animal studies. Catheter-based technique was used in three series of experiment in 7 sheep. First acute study was designed for evaluation if the screwing mode of guide wire anchoring to the right ventricular apex is feasible for the whole REMOT body fixing. Longer study was aimed to evaluate stability of the REMOT body in desired position when fixing the screwing wire on its both ends (to the right ventricular apex and to the skin in the neck area). X-ray methods and various morphological methods were used. The third acute study was intended to the REMOT body deployment without any fixing wire. In all of 7 sheep the REMOT was successfully inserted into the right heart cavities and then fixed to the right ventricular apex area. When the REMOT was left in situ more than 6 months it was stable, induced adhesion to the tricuspid valve leaflet and was associated with a specific cell invasion. Releasing of the REMOT from the guiding tools was also successfully verified. Deployment of the obturator body in the aim to reduce the tricuspid valve orifice is feasible and well tolerated in the short and longer term animal model. Specific cell colonization including neovascularization of the obturator body was observed.

Does folic acid supplementation rescue defects in ECE-1-deficient mouse embryos?

Endothelin (ET) signalling is essential for normal embryonic development. Disruption of this pathway leads to defects in the development of subsets of cranial and cephalic neural crest derivatives. Endothelin-converting enzyme 1 (ECE-1) is a ratelimiting step in the biosynthesis of ET-1. Recently, there has been considerable interest in the protective role of folic acid (FA) against congenital anomalies via increasing the expression of ET-1. We have tested whether FA supplementation can rescue craniofacial and cardiac defects observed in the ECE1-/- embryos. ECE1+/- mice were caged together to obtain litters containing embryos of all possible genotypes. The treatment group had the diet supplemented with 20 mg/kg of FA from the day of discovery of the vaginal plug. FA supplementation did not result in modified proportions of the genotypes, indicating no rescue of the embryonic mortality. There was also no effect on the litter size. Craniofacial and cardiac defects were likewise identical in the ECE1-/- embryos of both groups. There was a mild but significant reduction in the embryo size in wild-type and heterozygous FA-supplemented embryos, and there were haemorrhages in the wild-type supplemented embryos at ED14.5. Expression of ET receptor A detected by immunohistochemistry was up-regulated in the ECE1-/- embryos, but FA supplementation had no effects on the distribution of staining intensity. We conclude that FA is not able to rescue the phenotype in this model, suggesting an alternative pathway for its action. These results also caution against indiscriminate use of dietary supplements in attempts to prevent congenital anomalies.

Microarray analysis of normal and abnormal chick ventricular myocardial development.

The left and right ventricle originate from distinct parts of the cardiac tube, and several genes are known to be differentially expressed in these compartments. The aims of this study were to determine developmental differences in gene expression between the left and right ventricle, and to assess the effect of altered hemodynamic loading. RNA was extracted from isolated left and right normal chick embryonic ventricles at embryonic day 6, 8, and 10, and from day 8 left atrial ligated hearts with hypoplastic left and dilated right ventricles. cRNA was hybridized to Affymetrix Chicken Genome array according to manufacturer protocols. Microarray analysis identified 302 transcripts that were differentially expressed between the left and right ventricle. Comparative analysis detected 91 genes that were different in left ventricles of ligated hearts compared to age-matched ventricles, while 66 were different in the right ones. A large number of the changes could be interpreted as a delay of normal maturation. The approach described in this study could be used as one of the measures to gauge success of surgical procedures for congenital heart disease and help in determining the optimal time frame for intervention to prevent onset of irreversible changes.

Bendiocarbamate toxicity in the chick embryo.

Carbamate pesticides generally possess low toxicity for warm-blooded vertebrates, but developmental data are scarce. We have therefore evaluated embryotoxicity of choline esterase inhibitor bendiocarbamate in the chick embryo. The pesticide was dissolved in 5% acetone in distilled water and a volume of 200 microl was administered over the embryo through membrana papyracea on embryonic days 2, 3, 4, 5, and 10. Sampling was performed on embryonic day 10, while the embryos treated on embryonic day 10 were sampled on embryonic day 17. The toxicity of bendiocarbamate was fairly low, and LD50 decreased with advancing development from 1 mg/ embryo on embryonic day 2 to 29 mg on embryonic day 5. Malformations in surviving embryos were observed rarely (< 3 %) and occurred in both control and experimental groups. There was a mild but statistically significant dose-dependent reduction in body weight, most pronounced in the treatment on embryonic days 5 and 10, but the maximum difference from controls was below 15 %. A small but not significant increase in the number of positive cells was observed in the eye, limb buds, and the central nervous system of embryos treated on embryonic days 3 and 4 and examined after supravital whole-mount staining with Lysotracker Red for apoptosis. In agreement with previously published studies in other vertebrate animals, we conclude that bendiocarbamate does not possess significant toxicity in the avian embryo.

[Functional and developmental view on Purkinje fibers system]. / Funkcní a vývojový pohled na systém Purkynových vláken.

The cardiac conducting system is vital for generating and synchronizing the heartbeat. Beginning with Tawara, Einthoven and other pioneering workers, a wealth of information has been collected over the last 100 years on the histologic, morphologic and physiologic characteristics of specialized cardiac tissues. However, in the last ten years considerable effort has been put into understanding the cellular and molecular mechanisms governing its development. During this latter period, controversies have also arisen as to the nature of the signaling mechanisms involved in induction and patterning of the conducting system, particularly with respect to the pathways functioning in mammals. In this review, we will try to summarize the current state of knowledge in this field and point out some of the remaining questions.

Pacing redistributes glycogen within the developing myocardium.

Electrical pacing at physiological rate induces myocardial remodeling associated with regional changes in workload, blood flow and oxygen consumption. However, to what extent energy-producing pathways are also modified within the paced heart remains to be investigated. Pacing could particularly affect glycogen metabolism since hypertrophy stimulates glycolysis and increased workload favors glucose over fat oxidation. In order to test this hypothesis, we used the embryonic chick heart model in which ventricular pacing rapidly resulted in thinning of the ventricle wall and thickening of the atrial wall. Hearts of stage 22HH chick embryos were submitted in ovo to asynchronous and intermittent ventricular pacing delivered at physiological rate during 24 h. The resulting alterations of glycogen content were determined in atrium, ventricle and conotruncus of paced and sham-operated hearts. Hemodynamic parameters of the paced and spontaneously beating hearts were derived from computerized image analysis of video recordings. With respect to sham, paced hearts showed a significant decrease in glycogen content (nmoles glucose units/microg protein; mean+/-S.D.) only in atrium (1.48+/-0.40 v 0.84+/-0.34, n=8) and conotruncus (0.75+/-0.28 v 0.42+/-0.23, n=8). Pacing decreased the end diastolic and stroke volumes by 34 and 44%, respectively. Thus, the rapid glycogen depletion in regions remote from the stimulation site appears to be associated with regional changes in workload and remodeling. These findings underscore the importance of the coupling mechanisms between metabolic pathways and myocardial remodeling in the ectopically paced heart.

Structure and function of the developing zebrafish heart.

The combination of optical clarity and large scale of mutants makes the zebrafish vital for developmental biologists. However, there is no comprehensive reference of morphology and function for this animal. Since study of gene expression must be integrated with structure and function, we undertook a longitudinal study to define the cardiac morphology and physiology of the developing zebrafish. Our studies included 48-hr, 5-day, 2-week, 4-week, and 3-month post-fertilization zebrafish. We measured ventricular and body wet weights, and performed morphologic analysis on the heart with H&E and MF-20 antibody sections. Ventricular and dorsal aortic pressures were measured with a servonull system. Ventricular and body weight increased geometrically with development, but at different rates. Ventricle-to-body ratio decreased from 0.11 at 48-hr to 0.02 in adult. The heart is partitioned into sinus venosus, atrium, ventricle, and bulbus arteriosus as identified by the constriction between the segments at 48-hr. Valves were formed at 5-day post-fertilization. Until maturity, the atrium showed extensive pectinate muscles, and the atrial wall increased to two to three cell layers. The ventricular wall and the compact layer increased to three to four cell layers, while the extent and complexity in trabeculation continued. Further thickening of the heart wall was mainly by increase in cell size. The bulbus arteriosus had similar characteristics to the myocardium in early stages, but lost the MF-20 positive staining, and transitioned to smooth muscle layer. All pressures increased geometrically with development, and were linearly related to stage-specific values for body weight (P < 0.05). These data define the parameters of normal cardiac morphology and ventricular function in the developing zebrafish.

Developmental patterning of the myocardium.

The heart in higher vertebrates develops from a simple tube into a complex organ with four chambers specialized for efficient pumping at pressure. During this period, there is a concomitant change in the level of myocardial organization. One important event is the emergence of trabeculations in the luminal layers of the ventricles, a feature which enables the myocardium to increase its mass in the absence of any discrete coronary circulation. In subsequent development, this trabecular layer becomes solidified in its deeper part, thus increasing the compact component of the ventricular myocardium. The remaining layer adjacent to the ventricular lumen retains its trabeculations, with patterns which are both ventricle- and species-specific. During ontogenesis, the compact layer is initially only a few cells thick, but gradually develops a multilayered spiral architecture. A similar process can be charted in the atrial myocardium, where the luminal trabeculations become the pectinate muscles. Their extent then provides the best guide for distinguishing intrinsically the morphologically right from the left atrium. We review the variations of these processes during the development of the human heart and hearts from commonly used laboratory species (chick, mouse, and rat). Comparison with hearts from lower vertebrates is also provided. Despite some variations, such as the final pattern of papillary or pectinate muscles, the hearts observe the same biomechanical rules, and thus share many common points. The functional importance of myocardial organization is demonstrated by lethality of mouse mutants with perturbed myocardial architecture. We conclude that experimental studies uncovering the rules of myocardial assembly are relevant for the full understanding of development of the human heart.

Optimisation of the formation and distribution of protoporphyrin IX in the urothelium: an in vitro approach.

PURPOSE: To optimize conditions for photodynamic detection (PDD) and photodynamic therapy (PDT) of bladder carcinoma, urothelial accumulation of protoporphyrin IX (PpIX) and conditions leading to cell photodestruction were studied. MATERIALS AND METHODS: Porcine and human bladder mucosae were superfused with derivatives of 5-aminolevulinic acid (ALA). PpIX accumulation and distribution across the mucosa was studied by microspectrofluorometry. Cell viability and structural integrity were assessed by using vital dyes and microscopy. RESULTS: ALA esters, especially hexyl-ALA, accelerated and regularized urothelial PpIX accumulation and allowed for necrosis upon illumination. CONCLUSIONS: hexyl-ALA used at micromolar concentrations is the most efficient PpIX precursor for PDD and PDT.

The chick embryo heart as an experimental setup for the assessment of myocardial remodeling induced by pacing.

The mechanisms regulating remodeling of the heart are not well understood and only rarely investigated for pacing. We therefore developed a model based on the well-established chick embryo heart preparation. Hamburger Hamilton 21 stage Leghorn chick embryos were used. Access to the heart was obtained after having dissected the shell membranes. The electrodes (platinum wires) were placed in ovo: the anode on the vitelline membrane and the cathode at different sites of the heart (sinus venosus, base/apex of the ventricle). Sensing and stimulation thresholds were measured. Survival of the paced chick was studied. Among 30 chick embryonic hearts, the stimulation thresholds were 1.4 mV +/- 0.5 SD for the atrium, 2.6 V +/- 1.4 SD at the base, and 3.2 V +/- 1.5 SD at the apex of the ventricle, while the sensing signals were 1.3 mV +/- 0.5 SD at the atrium, 19.6 mV +/- 4.1 SD at the base, and 21.6 mV +/- 3.9 SD at the apex of the ventricle. Continuous pacing (pacing rate = intrinsic rate + 10%) could be maintained for 1.5 hours +/- 0.5 SD at the atrium, 8.9 hours +/- 0.7 SD at the base of the ventricle, and 7.9 hours +/- 1 SD at the apex of the ventricle up to death of the embryos. By using intermittent electrical stimulation, the association of 5 minutes on/5 minutes off pattern during 18 hours and 5 minutes on/15 minutes off, during 30 hours resulted in an effective pacing period of 19 hours in 60% of the experiments, reflecting 15 cell turnover cycles. This experimental setup will allow the study of morphological, metabolic, and molecular bases of ventricular remodeling induced by electrical stimulation.

Pacing-induced ventricular remodeling in the chick embryonic heart.

Chronic ectopic pacing in the adult heart induces myocardial hypotrophy close to the pacing site. We have recently described a similar localized decrease of compact myocardium thickness in the chick embryonic heart after 48 h of intermittent apical ventricular pacing. Here we analyze the cellular mechanisms underlying the response of the embryonic heart to pacing. Because the developing heart had been found to adjust its morphology according to functional demands by undergoing cellular hyperplasia or hypoplasia, we hypothesized that the stimulation should result in hypoplasia of the apical ventricular compartment. Morphologic analysis of hearts submitted to 18 h of effective pacing during 48 h showed a mild to moderate ventricular dilatation, a 28% decrease in the apical compact layer thickness with no changes in other ventricular locations, and atrial wall thickening. These modifications were caused by changes in the number of cell layers, whereas cell size was similar between paced and control hearts. Analysis of proliferative activity after 24 h of pacing showed a decrease of 32% in the rate of cell proliferation limited to the apical compact layer exposed to stimulation. No ultrastructural injury or increased cell death was found. These changes were accompanied by down-regulation of the myocardial growth factor fibroblast growth factor-2 but no differences were found in the expression of platelet-derived growth factor. Thus, chronic intermittent ventricular pacing induces myocardial remodeling in the chick embryonic heart, on the basis of locally regulated rates of cell proliferation.

Remodeling of chick embryonic ventricular myoarchitecture under experimentally changed loading conditions.

Adult myocardium adapts to changing functional demands by hyper- or hypotrophy while the developing heart reacts by hyper- or hypoplasia. How embryonic myocardial architecture adjusts to experimentally altered loading is not known. We subjected the chick embryonic hearts to mechanically altered loading to study its influence upon ventricular myoarchitecture. Chick embryonic hearts were subjected to conotruncal banding (increased afterload model), or left atrial ligation or clipping, creating a combined model of increased preload in right ventricle and decreased preload in left ventricle. Modifications of myocardial architecture were studied by scanning electron microscopy and histology with morphometry. In the conotruncal banded group, there was a mild to moderate ventricular dilatation, thickening of the compact myocardium and trabeculae, and spiraling of trabecular course in the left ventricle. Right atrioventricular valve morphology was altered from normal muscular flap towards a bicuspid structure. Left atrial ligation or clipping resulted in hypoplasia of the left heart structures with compensatory overdevelopment on the right side. Hypoplastic left ventricle had decreased myocardial volume and showed accelerated trabecular compaction. Increased volume load in the right ventricle was compensated primarily by chamber dilatation with altered trabecular pattern, and by trabecular proliferation and thickening of the compact myocardium at the later stages. A ventricular septal defect was noted in all conotruncal banded, and 25% of left atrial ligated hearts. Increasing pressure load is a main stimulus for embryonic myocardial growth, while increased volume load is compensated primarily by dilatation. Adequate loading is important for normal cardiac morphogenesis and the development of typical myocardial patterns.

The role of cell death in limb development of rats manifesting Lx allele on different genetic backgrounds.

Control of vertebrate digital pattern is a phylogenetically old mechanism. Animal strains with abnormal digital counts are a useful model system to study tissue, cell and molecular factors involved in limb patterning. The aim of this study was to investigate rat limb morphogenesis on gestation days 13 to 16 in normodactylous, polydactylous and oligodactylous fetuses where the deviation from the normal pentadactylous phenotype is caused by interaction of mutant Lx allele with different genetic backgrounds. General development was assessed by measurements of crown-rump length, and limb morphogenesis by hand and foot plate width. Skeletogenesis was studied histologically and by whole mount staining with Alcian Blue and Acridine Orange. Cell death was demonstrated by supravital staining and fluorescence microscopy and by standard histology on serial sections. No phenotypic differences among the groups were noted on day 13. On day 14, the oligodactylous hind limb buds were more spiky than normal and had well-developed preaxial necrotic site (foyer preaxial primaire) which was normally observed only on day 15. This area of programmed cell death was severely attenuated in polydactylous limb buds. Pollex triphalangy manifested as increased hand plate width from day 15. Also hind limb buds width differed by this stage between groups. No acceleration or retardation of skeletogenesis was observed in abnormal limbs. The data confirm the crucial role of spatial and temporal patterns of morphogenetic programmed cell death in control of digital pattern.