Bradley B. Keller

Cellular changes in experimental left heart hypoplasia

Hypoplastic left heart syndrome (HLHS) is a rare but deadly congenital malformation, which can be created experimentally in the chick embryo by left atrial ligation (LAL). The goal of this study was to examine the cellular changes leading to the profound remodeling of ventricular myocardial architecture that occurs in this model. Hypoplasia of left heart structures was produced after 3H‐thymidine prelabeling by partial LAL at stage 24, thereby reducing its volume, and redistributing blood preferentially to the developing right ventricle (RV). Controls included both sham‐operated and intact stage‐matched embryos. Survivors were studied 4 days after the ligation, when the heart organogenesis was essentially complete. Paraffin sections of the hearts were subjected to autoradiography and immunohistochemistry to detect changes in history of cell proliferation and expression of myosin, and growth factors implicated in cardiomyocyte proliferation. Sampling for apoptosis detection using TUNEL assay was done at stages 29 and 34. LAL resulted in decreased levels of proliferation in the left ventricular compact layer and trabeculae. The right ventricular compact layer also showed a slight decrease, but the trabeculae showed no differences. Anti‐myosin staining was significantly reduced in all compartments. The expression levels of growth factors were altered as well. Apoptosis was increased in the right atrioventricular mesenchyme, with no changes in the working myocardium. These data suggest that changes in cardiomyocyte proliferation play a significant role in the pathogenesis of HLHS. Anat Rec 267:137–145, 2002.

Identification of the T-Type Calcium Channel (CaV3.1d) in Developing Mouse Heart

Abstract— During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca2+) channels are critical for maintaining both contractility and excitability. In the present study, we identified the CaV3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. CaV3.1d is a splice variant of the &agr;1G, T-type Ca2+ channel. Immunohistochemical localization showed expression of &agr;1G Ca2+ channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the &agr;1G channels. Dihydropyridine-resistant inward Ba2+ or Ca2+ currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the &agr;1G channel expressed in human embryonic kidney-293 cells. Ni2+ blockade discriminates among T-type Ca2+ channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca2+ handling proteins. We demonstrate that 100 &mgr;mol/L Ni2+ partially blocked &agr;1G currents under physiological external Ca2+. We conclude that &agr;1G T-type Ca2+ channels are functional in midgestational fetal myocardium.

In Vivo Assessment of Embryonic Cardiovascular Dimensions and Function in Day-10.5 to -14.5 Mouse Embryos

Embryonic cardiovascular function has been extensively studied in vivo in the chick embryo. However, the geometry of mammalian and avian hearts differs; the mammalian cardiovascular system is coupled to both yolk sac and placental circulations, and unique murine genetic models associated with structural and functional cardiovascular defects are now available. We therefore adapted techniques validated for the chick embryo to define cardiovascular dimensions and function in the mouse embryo. We bred C3HeB female and C57B1/J6 male mice and ICR pairs for experiments on embryonic days (EDs) 10.5 to 14.5 (n = 130 dams). After maternal anesthesia (pentobarbital, 60 mg/kg IP), laparotomy, and sequential regional hysterotomy, we exposed and then imaged individual embryos at 60 Hz (video) in the ventral and/or left anterior oblique views while maintaining uteroplacental continuity. We measured epicardial chamber dimensions and then calculated right and left ventricular elliptical volumes from ares. In addition, we measured pulsed-Doppler blood velocity across the atrioventricular cushions and ventricular outflow tract. We maintained embryonic temperature with a heated surgical platform, topical oxygenated and warmed buffer, and warming lamps. Embryonic heart rate increased from 124.7 +/- 5.2 to 194.3 +/- 13.2 bpm from EDs 10.5 to 14.5 (P < .01). Right and left ventricular end-diastolic and end-systolic dimensions increased (P < .05 by ANOVA for each). Maximal ventricular mean inflow and outflow velocities increased from 62.33 +/- 4.06 to 106.23 +/- 11.59 and from 55.79 +/- 6.11 to 91.61 +/- 6.93 mm/s, respectively (P < .05 by ANOVA for each). Thus, as has been done for chick and rat embryos, the maturation of murine embryonic cardiovascular function can be quantified in vivo, setting the stage for the investigation of structure-function relations in mouse models of cardiovascular development and disease.

Antioxidant levels represent a major determinant in the regenerative capacity of muscle stem cells.

Stem cells are classically defined by their multipotent, long-term proliferation, and self-renewal capabilities. Here, we show that increased antioxidant capacity represents an additional functional characteristic of muscle-derived stem cells (MDSCs). Seeking to understand the superior regenerative capacity of MDSCs compared with myoblasts in cardiac and skeletal muscle transplantation, our group hypothesized that survival of the oxidative and inflammatory stress inherent to transplantation may play an important role. Evidence of increased enzymatic and nonenzymatic antioxidant capacity of MDSCs were observed in terms of higher levels of superoxide dismutase and glutathione, which appears to confer a differentiation and survival advantage. Further when glutathione levels of the MDSCs are lowered to that of myoblasts, the transplantation advantage of MDSCs over myoblasts is lost when transplanted into both skeletal and cardiac muscles. These findings elucidate an important cause for the superior regenerative capacity of MDSCs, and provide functional evidence for the emerging role of antioxidant capacity as a critical property for MDSC survival post-transplantation.

Myogenic endothelial cells purified from human skeletal muscle improve cardiac function after transplantation into infarcted myocardium.

OBJECTIVES The aim of this study was to evaluate the therapeutic potential of human skeletal muscle-derived myoendothelial cells for myocardial infarct repair. BACKGROUND We have recently identified and purified a novel population of myoendothelial cells from human skeletal muscle. These cells coexpress myogenic and endothelial cell markers and produce robust muscle regeneration when injected into cardiotoxin-injured skeletal muscle. METHODS Myoendothelial cells were isolated from biopsies of human skeletal muscle using a fluorescence-activated cell sorter along with populations of regular myoblasts and endothelial cells. Acute myocardial infarction was induced in male immune-deficient mice, and cells were directly injected into the ischemic area. Cardiac function was assessed by echocardiography, and donor cell engraftment, angiogenesis, scar tissue, endogenous cardiomyocyte proliferation, and apoptosis were all evaluated by immunohistochemistry. RESULTS A greater improvement in left ventricular function was observed after intramyocardial injection of myoendothelial cells when compared with that seen in hearts injected with myoblast or endothelial cells. Transplanted myoendothelial cells generated robust engraftments within the infarcted myocardium, and also stimulated angiogenesis, attenuation of scar tissue, and proliferation and survival of endogenous cardiomyocytes more effectively than transplanted myoblasts or endothelial cells. CONCLUSIONS Our findings suggest that myoendothelial cells represent a novel cell population from human skeletal muscle that may hold promise for cardiac repair.

Residual strain in the ventricle of the stage 16-24 chick embryo.

Residual stress and strain, i.e., the stress and strain remaining in a solid when all external loads are removed, may be produced in biological tissues by differential growth. During cardiac development, residual stress and strain may play a role in cardiac morphogenesis by affecting ventricular wall stress. After a transmural radial cut, a passive ventricular cross section opens into a sector, and the size of the opening angle provides a measure of the circumferential residual strain. Residual strains were characterized in this manner for the apical region of the diastolic embryonic chick heart for Hamburger-Hamilton stages 16, 18, 21, and 24 (approximately 2.5, 3.5, 4.0, and 4.5 days, respectively, of a 21-day incubation period). The average opening angle at these stages was 107 +/- 10 degrees, 79 +/- 10 degrees, 73 +/- 11 degrees, and 74 +/- 7 degrees, respectively (n > or = 5 for each stage). These measured angles were correlated with changes in ventricular morphology. Scanning electron micrographs of the apex revealed that the wall of the ventricle is smooth at stage 16. Then at stage 18, myocardial trabeculae develop, forming ridges with primarily a circumferential orientation. By stage 21, the trabeculae develop into a mesh, giving the ventricular wall a spongelike appearance, and the preferred orientation is lost by stage 24. The large decrease in opening angle between stages 16 and 18 corresponded to the onset of trabeculation, which is the greatest change in form during the studied stages. We speculate that residual strain is an important biomechanical factor during cardiac morphogenesis.

Development of cardiovascular systems : molecules to organisms

List of contributors Foreword Constance Weinstein Introduction. Why study cardiovascular development? Warren B. Burggren and Bradley B. Keller Part I. Molecular Cellular and Integrative Mechanisms: Determining Cardiovascular Development: 1. Genetic dissection of heart development Jau-Nian Chen and Mark C. Fishman 2. Cardiac membrane characteristics Lynn Mahony 3. Development of the myocardial contractile system Anne M. Murphy 4. Vasculogenesis and angiogenesis of the developing heart Robert J. Tomanek and Anna Ratajska 5. Extracellular matrix maturation and morphogenesis Wayne Carver, Louis Terracio and Thomas K. Borg 6. Endothelial maturation Jackson Wong 7. Embryonic cardiovascular function, coupling and maturation: a species view Bradley B. Keller 8. Hormonal systems regulating the cardiovascular system Makoto Nakazawa and Fusae Kajio Part II. Species Diversity in Cardiovascular Development: 9. Evolution of cardiovascular systems: insights into ontogeny Anthony P. Farrell 10. Morphogenesis of vertebrate hearts Jose M. Icardo 11. Invertebrate cardiovascular development Brian R. McMahon, George B. Bourne and Ka Hou Chu 12. Piscine cardiovascular development Peter J. Rombough 13. Amphibian cardiovascular development Warren W. Rurggren and Regina Fritsche 14. Reptilian cardiovascular development Stephen J. Warburton 15. Avian cardiovascular development Hiroshi Tazawa and Ping-Chun Lucy Hou 16. Mammalian cardiovascular development Kent L. Thornburg, George D. Giraud, Mark D. Reller and Mark J. Morton Part III. Environment and Disease in Cardiovascular Development: 17. Oxygen, temperature and pH influences on the development of non-mammalian embryos and larvae Bernd Pelster 18. Modelling gas exchange in embryos, larvae and fetuses Alan W. Pinder 19. Principles of abnormal cardiac development Adriana C. Gittenberger-de Groot and Robert E. Poelmann 20. In utero and postnatal interventions in cardiovascular malformations V. Mohan Reddy and Frank L. Hanley 21. Insights into the future care of congenital cardiovascular malformations Edward B. Clark Epilogue. Future directions in developmental cardiovascular sciences Bradley B. Keller and Warren W. Burggren References Systematic index Subject index.

Cellular antioxidant levels influence muscle stem cell therapy

Although cellular transplantation has been shown to promote improvements in cardiac function following injury, poor cell survival following transplantation continues to limit the efficacy of this therapy. We have previously observed that transplantation of muscle-derived stem cells (MDSCs) improves cardiac function in an acute murine model of myocardial infarction to a greater extent than myoblasts. This improved regenerative capacity of MDSCs is linked to their increased level of antioxidants such as glutathione (GSH) and superoxide dismutase. In the current study, we demonstrated the pivotal role of antioxidant levels on MDSCs survival and cardiac functional recovery by either reducing the antioxidant levels with diethyl maleate or increasing antioxidant levels with N-acetylcysteine (NAC). Both the anti- and pro-oxidant treatments dramatically influenced the survival of the MDSCs in vitro. When NAC-treated MDSCs were transplanted into infarcted myocardium, we observed significantly improved cardiac function, decreased scar tissue formation, and increased numbers of CD31(+) endothelial cell structures, compared to the injection of untreated and diethyl maleate-treated cells. These results indicate that elevating the levels of antioxidants in MDSCs with NAC can significantly influence their tissue regeneration capacity.

Morphological and mechanical characteristics of the reconstructed rat abdominal wall following use of a wet electrospun biodegradable polyurethane elastomer scaffold

Although a variety of materials are currently used for abdominal wall repair, general complications encountered include herniation, infection, and mechanical mismatch with native tissue. An approach wherein a degradable synthetic material is ultimately replaced by tissue mechanically approximating the native state could obviate these complications. We report here on the generation of biodegradable scaffolds for abdominal wall replacement using a wet electrospinning technique in which fibers of a biodegradable elastomer, poly(ester urethane)urea (PEUU), were concurrently deposited with electrosprayed serum-based culture medium. Wet electrospun PEUU (wet ePEUU) was found to exhibit markedly different mechanical behavior and to possess an altered microstructure relative to dry processed ePEUU. In a rat model for abdominal wall replacement, wet ePEUU scaffolds (1x2.5 cm) provided a healing result that developed toward approximating physiologic mechanical behavior at 8 weeks. An extensive cellular infiltrate possessing contractile smooth muscle markers was observed together with extensive extracellular matrix (collagens, elastin) elaboration. Control implants of dry ePEUU and expanded polytetrafluoroethylene did not experience substantial cellular infiltration and did not take on the native mechanical anisotropy of the rat abdominal wall. These results illustrate the markedly different in vivo behavior observed with this newly reported wet electrospinning process, offering a potentially useful refinement of an increasingly common biomaterial processing technique.

Thresholds for premature ventricular contractions in frog hearts exposed to lithotripter fields

Piezoelectrically generated lithotripter shocks were shown to produce premature ventricular contractions of the frog heart. Anesthetized grass frogs, Rana pipiens, were studied following implantation of an aortic catheter and EKG leads. The most sensitive phase of the heart cycle for the generation of premature ventricular contractions with lithotripter shocks at 30 MPa peak pressure was found to be the T-P segment. During this phase of the heart cycle, the minimum peak-positive pressure shock wave necessary to produce a premature ventricular contraction in a frog heart was between 5 MPa and 10 MPa.