Faculty Bibliography

Chronic hypertension, known to affect the collagen profile of the heart, and exercise result in impaired or improved heart function, respectively. Collagen types I [alpha 1(I)2 and alpha 2(I)] and III [alpha 1(III)3] are the predominant interstitial collagens thought to influence cardiac function, and the ratio of type III to I (collagen III/I) is thought to be a significant factor in the altered relaxation observed in hypertrophy. The present study tested the hypothesis that the myocardial structure and function are different in chronically exercise-trained vs. hypertensive rat hearts. Male rats were either chronically exercised (XTr) or submitted to experimental hypertension by coarctation of the abdominal aorta (Hyp) for 10 wks. Heart rate, blood pressure, and maximal rate of fall of the left ventricular pressure (-dp/dt) were recorded during isoproterenol stimulation. Results showed that both Hyp and XTr had higher heart weight and left ventricular weight-to-body weight ratios (P < 0.05). Mean arterial pressure (MAP) was higher in Hyp and lower in XTr (P < 0.05), whereas (-dP/dt)/MAP was diminished in Hyp but enhanced in XTr. Left ventricular collagen was higher in Hyp than XTr, whereas collagen III/I was reduced in Hyp compared with XTr (P < 0.05). Scanning and transmission electron microscopy also supported an accumulation of left ventricular collagen in Hyp compared with XTr. A negative correlation was observed between collagen III/I and (-dP/dt)/ MAP (r = -0.91; P < 0.05). These results suggest an important relationship between adaptations in left ventricular collagen and the changes in diastolic function observed in both chronic hypertension and exercise cardiac stress.

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the beta 1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-beta, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the alpha 2 beta 1 and, to a lesser extent, the alpha 1 beta 1 integrin complexes. The present studies were undertaken to determine the role of the alpha 1 integrin chain, a collagen/laminin receptor, in collagen gel contraction by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the alpha 1 beta 1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the alpha 1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the alpha 1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the alpha 1 beta 1 integrin complex plays a significant role in cellular interactions with interstitial collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing.

The excitation-contraction coupling cycle (ECC) consists of a complex cascade of electrochemical and mechanical events; however, the relative contributions of these different processes in the regulation of cardiac myofibrillar structure are not well understood. There is extensive evidence to suggest that the mechanical aspects of the ECC play a crucial role in controlling the availability of contractile proteins for myofibrillar assembly. To examine if these physical forces might also serve to stabilize the structure of preexisting myofibrils, beating and nonbeating cultures of neonatal cardiac myocytes (NCM) were subjected to a 5% static stretch. Contractile arrest was achieved by treating NCM with 12 microM nifedipine, which resulted in immediate and sustained contractile arrest and initiated the evolution of marked myofibrillar abnormalities within 24 hours. As judged by scanning confocal and transmission electron microscopic examination, an external load appears to partially stabilize myofibrillar structure in nonbeating NCM. These results suggest that the maintenance of myofibrillar structure may be highly dependent upon the mechanical aspects of ECC.

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

Cell-cell and cell-matrix interactions play critical roles in various developmental processes including differentiation, proliferation, and migration. Members of the integrin family of cell surface components are important mediators of these cell-extracellular matrix (ECM) contacts or interactions. The ECM provides signals to individual cells essential for development and differentiation and plays essential roles in establishing and maintaining the complex structure of the vertebrate heart. Integrins provide a fundamental link for transduction of developmental signals to cells. Integrin expression by cardiac myocytes is altered during neonatal heart development and disease; however, little is known regarding the spatial and temporal patterns of integrin expression during embryonic and fetal heart development. Essential to understanding the role of integrins in the organization of the heart, the present studies have localized beta-1 integrin protein and mRNA in fetal and neonatal rat hearts. Beta-1 integrin is predominantly found in regions of remodeling (trabeculae) in the early heart (10-13 days of gestation). Later in development (15 days of gestation onward), beta-1 integrin is abundant in regions containing an elaborate ECM, such as the valves. These studies further support the hypothesis that the expressions of integrins and ECM are coordinately regulated in the developing heart.

Secretion of the antidiuretic hormone (ADH) vasopressin is increased when body fluid homeostasis is disturbed by dehydration. Associated with this increased secretion is an elevation of vasopressin mRNA in magnocellular hypothalamic neurons projecting to the posterior pituitary. The proto-oncogene c-fos codes for a nuclear phospho-protein Fos which binds to specific DNA elements and acts as a transcriptional regulator coupling short-term extracellular stimuli to long-term responses by altering secondary target gene expression. This study in rats examined the time courses of dehydration induced c-fos expression and the change of vasopressin gene expression in the magnocellular neurons of the hypothalamus. Immunocytochemical and in situ hybridization study demonstrated that c-fos was induced by acute intracellular dehydration in the hypothalamic magnocellular nuclei of paraventricular (PVN), supraoptic (SON), and accessory groups such as nucleus circularis. Double-label immunocytochemical study co-localized Fos and vasopressin-neurophysin immunoreactivity in the same magnocellular neurons in the SON and PVN. In situ hybridization analysis after acute dehydration revealed a rapid and transient c-fos induction followed by a persistent increase in vasopressin mRNA for up to 2 days even after rehydration. Furthermore, prevention of c-fos translation by pretreatment with protein synthesis inhibitor cycloheximide attenuated this dehydration induced increase in vasopressin mRNA. This study demonstrated that an increase in vasopressin transcription after acute dehydration is dependent on an early phase of protein synthesis.

Angiotensin II (Ang II), a vasoactive octapeptide, has been implicated in cardiac growth and the development of hypertrophy and fibrosis secondary in hypertensive disease. These consequences of Ang II imply an effect on the function and morphology of cardiac interstitial cells (fibroblasts). The present investigation was designed to (1) determine whether neonatal heart fibroblasts (NHFs) possess functional Ang II receptors on their plasma membrane and (2) examine the effects of Ang II on NHFs in vitro using three- and two-dimensional (3D and 2D, respectively) cultures. Several analytic techniques were used to test the specific questions of the present study. Since cardiac fibroblast phenotype can be influenced by culture conditions, both 2D and 3D cultures were used in the present investigations. Reverse-transcriptase polymerase chain reaction and radioligand binding analysis were used to test for the presence of Ang II receptors on NHFs. Both revealed that NHFs in 2D culture possess Ang II receptor mRNA and Ang II receptors. When isolated NHFs were cultured in 3D collagen gels and treated with Ang II, gel contraction was stimulated by NHFs. This effect was attenuated by the specific Ang II receptor antagonist [Sar1,Ala8]Ang II. Ang II-stimulated gel contraction was completely inhibited by extracellular matrix receptor (beta 1-integrin) antibodies (P < .05), supporting previous studies indicating that collagen gel contraction is mediated via the integrins. Immunofluorescent staining was used to test the localization of cell-surface integrins. A more intense staining pattern for beta 1-integrin in Ang II-treated versus control cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

beta 1 Integrins are a family of structurally related heterodimeric cell surface receptors that are involved in adhesion to molecules in the extracellular matrix (ECM) such as laminin (LN), fibronectin (FN), and collagen. These receptors are expressed by many cell types and mediate a variety of processes such as cell-matrix and cell-to-cell adhesion, cell migration, growth, and differentiation. The purpose of these studies was to identify and partially characterize beta 1 integrins on adenohypophyseal cells and to begin to elucidate their functional importance. Adenohypophyses were removed from adult male rats, dispersed using 0.25% trypsin, rinsed, and resuspended in a 1:1 mixture of Dulbecco's modified Eagle's medium and F12 medium containing 10% fetal bovine serum and antibiotics. Ten million cells were allowed to attach to each of five plastic culture dishes overnight. The next day, the adenohypophyseal cells were surface-labeled with 125I. The labeled cells were lysed and centrifuged. The supernatant was immunoprecipitated using preimmune IgGs (100 micrograms/ml) and was then incubated with a polyclonal antibody against the rat beta 1 family of integrins or with a variety of immune IgGs directed against the alpha subunit of the receptor (anti alpha 1, anti alpha 2, anti alpha 3, and anti alpha 5 antibodies). The receptors were then immunoprecipitated by addition of protein A-Sepharose or IgG1 Sepharose. After washing, the immunoprecipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Cultured adenohypophyseal cells expressed the beta 1 integrin subunit, which was associated with the alpha 1, alpha 2, alpha 3, and alpha 5 integrin subunits.(ABSTRACT TRUNCATED AT 250 WORDS)