Faculty Bibliography

The role of angiotensin II (Ang II) in the early embryonic development of the heart has not been examined. We have used RT-PCR to identify mRNA for angiotensinogen, angiotensin-converting enzyme, and the Ang II AT1 and AT2 receptors in embryonic day 10.25 Sprague-Dawley rats, and have used confocal microscopy to localize the AT1 receptor to the greater curvature of the developing ventricle in these animals at embryonic days (ED) 9.25 and 10.25. The antibodies used in immunolocalization studies did not distinguish between the AT1a and AT1b receptor subtypes. In whole embryo culture, Ang II added to the culture media resulted in increased ventricular growth and myocyte hypertrophy when treated embryos were compared to cultured littermate controls. Use of Losartan and PD123,319 to block the Ang II AT1 and AT2 receptors resulted in reduced ventricular development and cardiac dilation when compared to control and Ang II-treated embryos. Addition of Ang II and PD123,319 to the culture media also resulted in cardiac loop inversions which may be associated with disruption of normal myofibrillar development. These results clearly indicate an important role for Ang II in the early embryonic development of the heart.

-To determine whether the formation and maintenance of focal adhesions and costameres in cardiac myocytes are influenced by the mechanical forces that they transmit, we mechanically unloaded these cells by inhibiting their spontaneous contractile activity with the calcium-channel blocker nifedipine (12 μM). Interferencereflection and fluorescence microscopy revealed that within 24 h of arrest, β1-integrin- and vinculin-positive focal adhesions and costameres were disrupted. Loss of mature β1-integrin from the cell surface was observed in cell surfacelabeling experiments and in Western blots. Subjecting nonbeating cells to a 5% static stretch for 24 h resulted in an increase of 21% for β1-integrin and 39% for vinculin. Stretching beating cells resulted in 71 and 9% increases, respectively. Intracellular concentrations of pre-β1 were not affected by contractile activity or by stretch. Our results indicate that mechanical forces stabilize the cellular levels of β1-integrin and vinculin by possibly regulating their association with the formation and maintenance of focal adhesions and costameres. β1-integrin; vinculin

Regulation of the turnover of extracellular matrix (ECM) components has been attributed in part to matrix metalloproteases (MMP). Isolated cardiac myocytes and fibroblasts from different developmental stages express different patterns of MMPs in vitro. Zymography of media and cell extracts of fibroblasts and myocytes indicated several apparent molecular weights (Mr) with gelatinolytic activity with prominent bands at 92 and 72 kDa. No caseinolytic activity was detected. These MMPs were characteristic of known MMP-2 and MMP-9. Fibroblasts predominantly expressed the latent 72-kDa MMP, whereas myocytes expressed a latent 92-kDa MMP. Expression of these MMPs was not affected by density of culture or the type of ECM substrate on which the cells were grown. Sodium dodecyl sulfate (SDS)-activated MMP-2 showed specific cleavage patterns on collagen types I and III but not on fibronectin, collagen type IV, or laminin. The reaction of SDS-activated MMP-2 produced a 140-kDa fragment from collagen types I and III. No specific substrate patterns were observed with activated MMP-9. MMP-2 from fibroblasts could also be activated by mechanical tension developed by fibroblasts within collagen gels or by cyclically stretching Silastic membranes on which the fibroblasts were grown. When mechanical tension was inhibited in collagen gels by antibodies against the β1 integrin, the 72-kDa MMP, or cytochalasin D, the activated band at 62 kDa was not detected. Immunocytochemical localization with antibodies against MMP-2 showed a weak reaction on cardiac myocytes, but intense staining around the focal adhesions of migrating fibroblasts. In collagen gels, staining was localized to the leading pseudopodia of the fibroblasts. Together, these data indicate that the rat MMP-2 is a collagenase primarily associated with cardiac fibroblasts, activated by mechanical tension, and may be important in cellular ECM interactions.

To determine whether the formation and maintenance of focal adhesions and costameres in cardiac myocytes are influenced by the mechanical forces that they transmit, we mechanically unloaded these cells by inhibiting their spontaneous contractile activity with the calcium-channel blocker nifedipine (12 microM). Interference-reflection and fluorescence microscopy revealed that within 24 h of arrest, beta 1-integrin- and vinculin-positive focal adhesions and costameres were disrupted. Loss of mature beta 1-integrin from the cell surface was observed in cell surface-labeling experiments and in Western blots. Subjecting nonbeating cells to a 5% static stretch for 24 h resulted in an increase of 21% for beta 1-integrin and 39% for vinculin. Stretching beating cells resulted in 71 and 9% increases, respectively. Intracellular concentrations of pre-beta 1 were not affected by contractile activity or by stretch. Our results indicate that mechanical forces stabilize the cellular levels of beta 1-integrin and vinculin by possibly regulating their association with the formation and maintenance of focal adhesions and costameres.

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5+/-26.8% and 48.7+/-20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2+/-14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.

The long-range goal of this research is to establish an in vitro system that will permit pertubation of mammalian heart development and in situ examination of the cellular and molecular events underlying cardiac morphogenesis. Rat embryos at 9.5-11.5 days of gestation were placed in culture bottles containing rat serum and Tyrode's solution. Embryos cultured for 24 and 48 h were compared to age-matched in vivo controls for morphological score, morphometric analysis of heart development, and confocal and electron microscopic analysis of myofiber pattern formation. Morphological scores indicated that embryos cultured for 24 h from day 9.5 to 10.5 had essentially normal development when compared to age-matched embryos allowed to develop in vivo. Development of embryos maintained for 48 h in culture was slightly delayed at 66-68% of age matched in vivo embryos. Analysis of hearts from embryos allowed to develop 9.5-11.5 days in vivo plus 24 and 48 h in culture showed that the ventricular thickness and height, as well as the truncal, atrial and ventricular diameters were equivalent to those of hearts from age-matched in vivo controls. Hearts from embryos allowed to develop from 11.5-12.5 days in vitro and cultured for 24 and 48 h had smaller left ventricular and atrial dimensions than controls. Cardiac myofibrillogenesis and myofibrillar pattern formation in embryos cultured from 9.5 days of in vivo development for 48 h were also normal. These studies indicate that the rat whole embryo culture system is a useful model to study several critical periods in mammalian heart development.

We reported previously that coculture of immature rat Sertoli cells with Leydig cells or the addition of a concentrate from Sertoli cell-conditioned medium (SCCM) stimulated Leydig cell [(3)H]-thymidine incorporation, increased cell number, and altered Leydig cell morphology (Wu and Murono, 1994). In the present studies, the effect of various extracellular matrix proteins on immature Leydig cell binding, proliferation and response to SCCM concentrate was investigated. Pretreatment of culture wells with 50 μg/mL collagen I or 10 μg/mL laminin inhibited Leydig cell binding to culture wells about 95 and 89%, respectively; however, 5 μg/mL fibronectin did not change the level of attachment. The binding of Leydig cells to fibronectin was reduced by antifibronectin or-β1 integrin antibodies (66 and 91%, respectively). Treatment of culture wells with five or 50 μg/mL fibronectin alone increased [(3)H]thymidine incorporation about twofold. When Leydig cells were cultured in wells precoated with increasing concentrations of fibronectin and then treated with SCCM concentrate for 2 d, [(3)H]-thymidine incorporation increased progressively with the concentration of fibronectin, beyond the levels observed with SCCM concentrate alone. This response was associated with increases in both Leydig cell number and labeling indices. When Leydig cells were cultured on fibronectin, their numbers increased by 3.7-and 5.1-fold following treatment with SCCM concentrates or coculture for 6 d, respectively; whereas, they increased 2.6- and 3.9-fold, respectively, when cultured on plastic. Labeling indices of Leydig cells cultured on plastic for 2 d and treated with SCCM or cocultured were 6.9 and 11.9%, respectively, while labeling indices of Leydig cells grown on fibronectin increased further to 17.6 and 26.3%, respectively. α5β1 integrin complexes and α5 integrin mRNA were expressed in Leydig cells, suggesting that binding to fibronectin may be mediated by α5β1 integrins, a fibronectin receptor. These results suggest that Leydig cell proliferation stimulated by a Sertoli cell-secreted mitogenic factor(s) is enhanced by Leydig cell binding fibronectin, and that this binding may be mediated by α5β1 integrins.

BACKGROUND: In chickens, cytodifferentiation, right side dominance in myofibril development, and variations in myofibrillar patterns in different areas and layers of the myocardial wall exist which have been implicated in the process of heart looping. Little comparable information is available for developing myofibrillar patterns in the early development of mammalian hearts. METHODS: We have used transmission electron microscopy (TEM), confocal scanning laser microscopy (CSLM), and 3-D reconstruction techniques also present in the looping hearts of embryonic day (ED) 9.5 to 11.5 rat hearts. RESULTS: Local and regional variations and right side dominance in myofibrillar patterns were shown during looping in 9.5 through 11.5 days of development in embryonic rat heart. At 9.5 days of development, myofibrils near the lumen of the myocardial wall were primarily in circumferential bands while near the pericardial surface they were primarily in longitudinal bands. In older embryos, regional variations in myofibrillar organization was found in areas associated with the cardiac cushions, trabeculae, and myocardial wall of the developing heart chambers. Based on sarcomeric structure, myofibrils in the ventricle and outflow tract were more advanced than those found in the atrial wall. CONCLUSIONS: The local and regional patterns of myofibrils in looping rat hearts are similar to those which have been found in developing chicken hearts. This study and others indicate cytodifferentiation and development of the contractile apparatus has a crucial role in the process of heart looping.

Mechanical forces play an essential role in regulating the synthesis and assembly of contractile proteins into the sarcomeres of cardiac myocytes. To examine if physical forces might also regulate the turnover of contractile proteins at a posttranslational site of control, beating and nonbeating neonatal cardiac myocytes (NCM) were subjected to a 5% static stretch. The L-type calcium channel blocker nifedipine (12 microM) was used to inhibit contraction. Pulse-chase biosynthetic labeling experiments demonstrated that contractile arrest accelerated the loss of isotopic tracer from the total myofibrillar protein fraction, myosin heavy chain (MHC), and actin, but not desmin. Myofibrillar abnormalities developed in parallel with these metabolic changes. A 5% static load appeared to partially stabilize myofibrillar structure in nonbeating NCM and suppressed the loss of isotopic tracer from the total myofibrillar protein fraction, MHC, and actin in beating and nonbeating NCM. Contractile activity and/or a static stretch promoted the accumulation of MHC, actin, and desmin. Applying a static load to myocytes that lacked preexisting myofibrils did not promote the assembly of sarcomeres or alter protein turnover. These data indicate that the turnover of MHC and actin is correlated with the organizational state of the myofibrillar apparatus.

The cardiac interstitium represents a system of diverse extracellular matrix (ECM) components organized into a complex, three-dimensional network that surrounds the cellular components of the heart (Borg and Caulfield 1981, Weber et al. 1994, Comper 1995). The interaction of the cellular components with the interstitium is dynamic and occurs in response to physiological signals during development, normal homeostasis, and disease (Borg and Caulfield 1981, Weber et al. 1994). Both the quantitative and qualitative expression of ECM components play an important role in cardiac function; however, the mechanisms that regulate the expression and function are not well understood. The manner in which the terminally differentiated myocyte perceives its external environment is of critical importance to the function of the heart. These external signals are delivered via the other two major components of the heart: the vascular system and the surrounding interstitium or ECM. Although it is obvious that the vascular system provides the transport of a variety of regulatory components that influence the contractile ability of the myocyte, the role of the interstitium in relation to cardiac function is less understood and is the focus of this review.