Cardiac assist with a twist: apical torsion as a means to improve failing heart function

Changes in muscle fiber orientation across the wall of the left ventricle (LV) cause the apex of the heart to turn 10-15 deg in opposition to its base during systole and are believed to increase stroke volume and lower wall stress in healthy hearts. Studies show that cardiac torsion is sensitive to various disease states, which suggests that it may be an important aspect of cardiac function. Modern imaging techniques have sparked renewed interest in cardiac torsion dynamics, but no work has been done to determine whether mechanically augmented apical torsion can be used to restore function to failing hearts. In this report, we discuss the potential advantages of this approach and present evidence that turning the cardiac apex by mechanical means can displace a clinically significant volume of blood from failing hearts. Computational models of normal and reduced-function LVs were created to predict the effects of applied apical torsion on ventricular stroke work and wall stress. These same conditions were reproduced in anesthetized pigs with drug-induced heart failure using a custom apical torsion device programmed to rotate over various angles during cardiac systole. Simulations of applied 90 deg torsion in a prolate spheroidal computational model of a reduced-function pig heart produced significant increases in stroke work (25%) and stroke volume with reduced fiber stress in the epicardial region. These calculations were in substantial agreement with corresponding in vivo measurements. Specifically, the computer model predicted torsion-induced stroke volume increases from 13.1 to 14.4 mL (9.9%) while actual stroke volume in a pig heart of similar size and degree of dysfunction increased from 11.1 to 13.0 mL (17.1%). Likewise, peak LV pressures in the computer model rose from 85 to 95 mm Hg (11.7%) with torsion while maximum ventricular pressures in vivo increased in similar proportion, from 55 to 61 mm Hg (10.9%). These data suggest that: (a) the computer model of apical torsion developed for this work is a fair and accurate predictor of experimental outcomes, and (b) supra-physiologic apical torsion may be a viable means to boost cardiac output while avoiding blood contact that occurs with other assist methods.     

Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts

The concept of regenerating diseased myocardium by implantation of tissue-engineered heart muscle is intriguing, but convincing evidence is lacking that heart tissues can be generated at a size and with contractile properties that would lend considerable support to failing hearts. Here we created large (thickness/diameter, 1-4 mm/15 mm), force-generating engineered heart tissue from neonatal rat heart cells. Engineered heart tissue formed thick cardiac muscle layers when implanted on myocardial infarcts in immune-suppressed rats. When evaluated 28 d later, engineered heart tissue showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction. Moreover, engineered heart tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted hearts compared to controls (sham operation and noncontractile constructs). Thus, our study provides evidence that large contractile cardiac tissue grafts can be constructed in vitro, can survive after implantation and can support contractile function of infarcted hearts.     

A method for preparing freeze-clamped tissue samples for metabolite analyses

A rapid and simple method for preparing freeze-clamped tissue samples for metabolite determinations is described. Freeze-clamped rat heart tissue samples weighing from 0.8 to 1.0 g were homogenized directly in an Ultra-Turrax homogenizer for 60 s in 3.5 ml of ice-cold 0.6 M HClO4 without pulverizing them in liquid nitrogen. After centrifugation, the pellet was rehomogenized in the Ultra-Turrax homogenizer for 30 s in 1.5 ml of HClO4. Following a further centrifugation the extracts were combined and the pH was adjusted to 7.0 by adding 5 M K2CO3. The neutralized supernatant was used for the desired assays. The analyses of the tissue extracts obtained from isolated perfused rat hearts by the present method give similar results for different kinds of metabolites than those processed according to the previous classical method. Moreover, the values of the various parameters determined from the tissue extracts prepared according to the method described here are similar to the data reported in literature. The method can be readily applied to any other freeze-clamped tissue. The greatest improvement obtained is that the homogenization procedure can be accomplished easily and conveniently in about one-tenth of the time required for the earlier classical method without the time-consuming and unpleasant tissue grinding in liquid nitrogen.     

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.     

Cardiac Fine Structure in Selected Arthropods and Molluscs

The ultrastructure of the single-chambered hearts of selectedarthropods is compared with that of the multi-chambered heartsof three molluscs. I used the following four systems to makethe comparison: (1) contractile apparatus, (2) sarcoplasmicreticulum and surface invaginations, (3) cell to cell junctions,and (4) nerves. The contractile apparatus is composed of thinand thick filaments. While the thin filaments have the samediameter, the diameter of the thick filaments differs from oneheart to another. Evidence is presented to indicate that thisis due to varying amounts of paramyosin in the thick filaments.The arthropod cardiac cells have an extensive system of sarcoplasmicreticulum, the terminal vesicles of which are coupled to theplasmalemma and to the invaginations of the plasmalemma, theT-system. The molluscan cardiac cells lack a typical T-system,which is presumably due to their small cell size (about 10 µm).They possess, however, an elaborate system of sarcoplasmic reticulumwhich extends from just under the plasmalemma to the middleof the cell. In addition to elaborate sarcoplasmic reticulum,the heart of the whelk (Busycon canaliculatum) possess manysmall invaginations of the plasmalemma, called sarcolemmic tubules.These invaginations of the cell surface are not found in thehearts of the few bivalves examined. All arthropod and molluscanhearts have intercalated discs which can be seen in the lightmicroscope. Two types of junctions can be distinguished in theelectron microscope. The mechanical junction is at the levelof the terminal sarcomere where the thin filaments are embeddedin the cell wall and dense granular material appears to causethe two adjacent cells to adhere to each other. The electricaljunction is found along the lateral borders of cells of boththe molluscan and arthropod hearts. Finally, while nerves appearto be absent in the myogenic moth heart, they are abundant inthe myogenic cockroach heart and in the neurogenic lobster heart.Furthermore, two types of nerves appear very prominently inthe myogenic molluscan hearts.     

Stress Distribution in the Canine Left Ventricle during Diastole and Systole

A model is proposed for stress analysis of the left ventricular wall (LV wall) based on the realistic assumption that the myocardium is essentially composed of fiber elements which carry only axial tension and vary in orientation through the wall. Stress analysis based on such a model requires an extensive study of muscle fiber orientation and curvature through the myocardium. Accordingly, the principal curvatures were studied at a local site near the equator in ten dog hearts rapidly fixed in situ at end diastole and end systole; the fiber orientation for these hearts had already been established in a previous study. The principal radii of curvature were (a) measured by fitting templates to the endocardial and epicardial wall surfaces in the circumferential and longitudinal directions and (b) computed from measured lengths of semiaxes of ellipsoids of revolution representing the LV wall (“ellipsoid” data). The wall was regarded as a tethered set of nested shells, each having a unique fiber orientation. Results indicate the following. (a) Fiber curvature, k, is maximum at midwall at end systole; this peak shifts towards endocardium at end diastole. (b) The pressure or radial stress through the wall decreases more rapidly near the endocardium than near the epicardium at end diastole and at end systole when a constant tension is assumed for each fiber through the wall. (c) At end diastole the curve for the circumferential stress vs. wall thickness is convex with a maximum at midwall. In the longitudinal direction the stress distribution curve is concave with a minimum at midwall. Similar distributions are obtained at end systole when a constant tension is assumed for each fiber through the wall. (d) The curvature and stress distributions obtained by direct measurements at a selected local site agree well with those computed from “ellipsoid” data.     

Comparative stereometric study of the intramural arterial bed of the human myocardium using injection and non-injection methods]

Comparative sterometric study of intramural arterial bed of the left ventricle wall of 20 injected and 30 non-injected hearts of practically health adults aged from 24 to 27 years (after violent death) was conducted. The volumetric density index of intramural arterial bed(Vv) of the myocardium of the left ventricle wall was on the average 9 times less in case of non-injected hearts than in the injected ones. The Vv was the greastest in the middle type of the coronary circulation of the heart. The non-linear dependence between the Vv and the age was revealed. The Vv decreased with the increase of the heart weight. With the equal indices of the heart weight the Vv was significantly higher in females than in males. The non-injection sterometric method used for the study of the inorganic arterial bed simplifies and objectifies the estimation of the state of myocardial blood supply and can be used in anatomopathological practice.     

Myocardial fiber architecture in the human heart. Anatomical demonstration of modifications in the normal pattern of ventricular fiber architecture in a malformed adult specimen

Ventricular myocardial fiber architecture has been considered an important factor in heart dynamics. Most anatomical studies however have focussed on the analysis of normal hearts. The present study compares ventricular myocardial fiber architecture patterns in dissections of 5 normal hearts and a malformed human heart with membranous ventricular septal defect, overriding right aorta, pulmonic stenosis, with absent pulmonary valve and hypertrophied right ventricle. Qualitative and quantitative changes in ventricular myocardial fiber architecture were noted in the malformed heart.     

In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix

We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24-48 h and can be maintained in culture for 5-6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.     

Optimizing ventricular fibers: uniform strain or stress,but not ATP consumption,leads to high efficiency

The aim of this study was to investigate the influence of fiber orientation in the left ventricular (LV) wall on the ejection fraction, efficiency, and heterogeneity of the distributions of developed fiber stress, strain and ATP consumption. A finite element model of LV mechanics was used with active properties of the cardiac muscle described by the Huxley-type cross-bridge model. The computed variances of sarcomere length (SL(var)), developed stress (DS(var)), and ATP consumption (ATP(var)) have several minima at different transmural courses of helix fiber angle. We identified only one region in the used design space with high ejection fraction, high efficiency of the LV and relatively small SL(var), DS(var), and ATP(var). This region corresponds to the physiological distribution of the helix fiber angle in the LV wall. Transmural fiber angle can be predicted by minimizing SL(var) and DS(var), but not ATP(var). If ATP(var) was minimized, then the transverse fiber angle was considerably underestimated. The results suggest that ATP consumption distribution is not regulating the fiber orientation in the heart.     

Diffusion MRI Tractography of the Developing Human Fetal Heart

Objective

Human myocardium has a complex and anisotropic 3D fiber pattern. It remains unknown, however, when in fetal life this anisotropic pattern develops and whether the human heart is structurally fully mature at birth. We aimed here to use diffusion tensor MRI (DTI) tractography to characterize the evolution of fiber architecture in the developing human fetal heart.

Methods

Human fetal hearts (n = 5) between 10–19 weeks of gestation were studied. The heart from a 6-day old neonate and an adult human heart served as controls. The degree of myocardial anisotropy was measured by calculating the fractional anisotropy (FA) index. In addition, fiber tracts were created by numerically integrating the primary eigenvector field in the heart into coherent streamlines.

Results

At 10–14 weeks the fetal hearts were highly isotropic and few tracts could be resolved. Between 14–19 weeks the anisotropy seen in the adult heart began to develop. Coherent fiber tracts were well resolved by 19 weeks. The 19-week myocardium, however, remained weakly anisotropic with a low FA and no discernable sheet structure.

Conclusions

The human fetal heart remains highly isotropic until 14–19 weeks, at which time cardiomyocytes self-align into coherent tracts. This process lags 2–3 months behind the onset of cardiac contraction, which may be a prerequisite for cardiomyocyte maturation and alignment. No evidence of a connective tissue scaffold guiding this process could be identified by DTI. Maturation of the heart’s sheet structure occurs late in gestation and evolves further after birth.     

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.     

Microanatomy of the heart and associated structures of two scorpions,Centruroides sculpturatus ewing and Uroctonus mordax thorell

Spiroid orientation of the circumferential heart wall muscles is described for Centruroides sculpturatus Ewing. This muscle arrangement accounts for differences in ostial position when the heart of this species is compared to that of Uroctonus mordax Thorell. Other differences, such as number of lateral arteries present, cannot be explained on the basis of circumferential muscle orientation. The histology of the heart and associated vessels, but not the supraneural vessel, was found to be similar in both species. The lateral, posterior, communicating and sternal arteries all possess a muscularis composed of irregularly spaced, apparently branched, striated muscle fibers. External to this is a covering of connective tissue. The lumina of these arteries, the aorta, and the supraneural vessel are lined with a homogeneous, PAS-positive membrane. This membrane is also seen in blood vessels which penetrate the nervous system. It was not observed in vessels accompanying major nerves. Findings are compared to those of other authors. Differences in the structure of the hearts of these two species are discussed in relation to the microanatomy of other arachnid hearts.     

Functional Morphology of the Heart in Fishes

The systemic heart of fishes consists of four chambers in series,the sinus venosus, atrium, ventricle, and conus or bulbus. Valvesbetween the chambers and contraction of all chambers exceptthe bulbus maintain a unidirectional blood flow through theheart. The heart is composed of typical vertebrate cardiac muscle,although there may be minor differences in the distributionof spontaneously active cells, the rate and nature of spreadof excitatory waves, and the characteristics of resting andaction potentials between different fish and other vertebrates.Cholinergic fibers innervate the heart, except in hagfish whichhave aneural hearts. Fish hearts lack sympathetic innervation.The level of vagal tone varies considerably, and is affectedby many factors. In some fish the heart is essentially aneural(without vagal tone) during exercise and may resemble an isolatedmammalian ventricle with increased venous return causing increasedcardiac output. There are many mechanisms that could increasevenous return in exercising fish. rß-adrenergic receptorshave been located on the hearts of some fish, and changing levelsof catecholamines may play a role in regulating cardiac activity.Changes in cardiac output in fish are normally associated withlarge changes in stroke volume and small cha-nges in heart rate.     

Wall shear-rate estimation within the 50cc Penn State artificial heart using particle image velocimetry

Particle image velocimetry (PIV) has been gaining acceptance as a routine tool to evaluate the flow fields associated with fluid mechanical devices. We have developed algorithms to investigate the wall shear-rates within the 50cc Penn State artificial heart using low magnification, conventional particle image velocimetry (PIV). Wall shear has been implicated in clot formation, a major post-implant problem with artificial hearts. To address the issues of wall scattering and incomplete measurement volumes, associated with near wall measurements, we have introduced a zero masking and a fluid centroid shifting technique. Simulations using different velocity fields were conducted with the techniques to assess their viability. Subsequently, the techniques were applied to the experimental data collected. The results indicate that the size of the interrogation region should be chosen to be as small as possible to maximize resolution while large enough to ensure an adequate number of particles per region. In the current study, a 16 x 16 interrogation window performed well with good spatial resolution and particle density for the estimation of wall shear rate. The techniques developed with PIV allow wall shear-rate estimates to be obtained from a large number of sites at one time. Because a planar image of a flow field can be determined relatively rapidly, PIV may prove useful in any preliminary design procedure.     

Expression of cardiac neural crest and heart genes isolated by modified differential display

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.     

The low oxygen-carrying capacity of Krebs buffer causes a doubling in ventricular wall thickness in the isolated heart

The buffer-perfused Langendorff heart is significantly vasodilated compared with the in vivo heart. In this study, we employed ultrasound to determine if this vasodilation translated into changes in left ventricular wall thickness (LVWT), and if this effect persisted when these hearts were switched to the "working" mode. To investigate the effects of perfusion pressure, vascular tone, and oxygen availability on cardiac dimensions, we perfused hearts (from male Wistar rats) in the Langendorff mode at 80, 60, and 40 cm H2O pressure, and infused further groups of hearts with either the vasoconstrictor endothelin-1 (ET-1) or the blood substitute FC-43. Buffer perfusion induced a doubling in diastolic LVWT compared with the same hearts in vivo (5.4 +/- 0.2 mm vs. 2.6 +/- 0.2 mm, p < 0.05) that was not reversed by switching hearts to "working" mode. Perfusion pressures of 60 and 40 cm H2O resulted in an increase in diastolic LVWT. ET-1 infusion caused a dose-dependent decrease in diastolic LVWT (6.6 +/- 0.4 to 4.8 +/- 0.4 mm at a concentration of 10(-9) mol/L, p < 0.05), with a concurrent decrease in coronary flow. FC-43 decreased diastolic LVWT from 6.7 +/- 0.5 to 3.8 +/- 0.7 mm (p < 0.05), with coronary flow falling from 16.1 +/- 0.4 to 8.1 +/- 0.4 mL/min (p < 0.05). We conclude that the increased diastolic LVWT observed in buffer-perfused hearts is due to vasodilation induced by the low oxygen-carrying capacity of buffer compared with blood in vivo, and that the inotropic effect of ET-1 in the Langendorff heart may be the result of a reversal of this wall thickening. The implications of these findings are discussed.     

Lymph heart musculature is under distinct developmental control from lymphatic endothelium

Lymph hearts are pulsatile organs, present in lower vertebrates, that function to propel lymph into the venous system. Although they are absent in mammals, the initial veno-lymphatic plexus that forms during mammalian jugular lymph sac development has been described as the vestigial homologue of the nascent stage of ancestral anterior lymph hearts. Despite the widespread presence of lymph hearts among vertebrate species and their unique function, extremely little is known about lymph heart development. We show that Xenopus anterior lymph heart muscle expresses skeletal muscle markers such as myoD and 12/101, rather than cardiac markers. The onset of lymph heart myoblast induction can be visualized by engrailed-1 (en1) staining in anterior trunk somites, which is dependent on Hedgehog (Hh) signaling. In the absence of Hh signaling and upon en1 knockdown, lymph heart muscle fails to develop, despite the normal development of the lymphatic endothelium of the lymph heart, and embryos develop edema. These results suggest a mechanism for the evolutionary transition from anterior lymph hearts to jugular lymph sacs in mammals.