Steven A. Fisher

Apoptosis During Cardiovascular Development

Morphogenesis and developmental remodeling of cardiovascular tissues involve coordinated regulation of cell proliferation and apoptosis. In the heart, clear evidence points toward focal apoptosis as a contributor to development of the embryonic outflow tract, cardiac valves, conducting system, and the developing coronary vasculature. Apoptosis in the heart is likely regulated by survival and death signals that are also present in many other tissues. Cell type–specific regulation may be superimposed on general cell death/survival machinery through tissue-specific transcriptional pathways. In the vasculature, apoptosis almost certainly contributes to developmental vessel regression, and it is of proven importance in remodeling of arterial structure in response to local changes in hemodynamics. Physical forces, growth factors, and extracellular matrix drive vascular cell survival pathways, and considerable evidence points to local nitric oxide production as an important but complex regulator of vascular cell death. In both the heart and vasculature, progress has been impeded by inadequate information concerning the incidence of apoptosis, its relative importance compared with the diverse cell behaviors that remodel developing tissues, and by our primitive knowledge concerning regulation of cell death in these tissues. However, tools are now available to better understand apoptosis in normal and abnormal development of cardiovascular structures, and a framework has been established that should lead to considerable progress in the coming years.

The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs.

Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general has resulted in the development of new techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs.

Vascular smooth muscle phenotypic diversity and function.

The control of force production in vascular smooth muscle is critical to the normal regulation of blood flow and pressure, and altered regulation is common to diseases such as hypertension, heart failure, and ischemia. A great deal has been learned about imbalances in vasoconstrictor and vasodilator signals, e.g., angiotensin, endothelin, norepinephrine, and nitric oxide, that regulate vascular tone in normal and disease contexts. In contrast there has been limited study of how the phenotypic state of the vascular smooth muscle cell may influence the contractile response to these signaling pathways dependent upon the developmental, tissue-specific (vascular bed) or disease context. Smooth, skeletal, and cardiac muscle lineages are traditionally classified into fast or slow sublineages based on rates of contraction and relaxation, recognizing that this simple dichotomy vastly underrepresents muscle phenotypic diversity. A great deal has been learned about developmental specification of the striated muscle sublineages and their phenotypic interconversions in the mature animal under the control of mechanical load, neural input, and hormones. In contrast there has been relatively limited study of smooth muscle contractile phenotypic diversity. This is surprising given the number of diseases in which smooth muscle contractile dysfunction plays a key role. This review focuses on smooth muscle contractile phenotypic diversity in the vascular system, how it is generated, and how it may determine vascular function in developmental and disease contexts.

Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3′-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p < 0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.

Differential levels of tissue hypoxia in the developing chicken heart

Tissue hypoxia plays a critical role in normal development, including cardiogenesis. Previously, we showed that oxygen concentration, as assessed by the hypoxia indicator EF5, is lowest in the outflow tract (OFT) myocardium of the developing chicken heart and may be regulating events in OFT morphogenesis. In this study, we identified additional areas of the embryonic chicken heart that were intensely positive for EF5 within the myocardium in discrete regions of the atrial wall and the interventricular septum (IVS). The region of the IVS that is EF5‐positive includes a portion of the developing central conduction system identified by HNK‐1 co‐immunostaining. The EF5 positive tissues were also specifically positive for nuclear‐localized hypoxia inducible factor 1α (HIF‐1α), the oxygen‐sensitive component of the hypoxia inducible factor 1 (HIF‐1) heterodimer. The pattern of the most intensely EF5‐stained myocardial regions of the atria and IVS resemble the pattern of the major coronary vessels that form in later stages within or immediately adjacent to these particular regions. These vessels include the sinoatrial nodal artery that is a branch of the right coronary artery within the atrial wall and the anterior/posterior interventricular vessels of the IVS. These findings indicate that a portion of the developing central conduction system and the patterning of coronary vessels may be subject to a level of regulation that is dependent on differential oxygen concentration within cardiac tissues and subsequent HIF‐1 regulation of gene expression. Developmental Dynamics 235:115–123, 2006.

Initiation of apoptosis in the developing avian outflow tract myocardium.

Apoptosis occurs within the cardiac outflow tract (OFT) myocardium during normal development of chick hearts. This peak of apoptosis occurs at stage 30–31 and coincides with dramatic remodeling of the OFT, suggesting that apoptosis occurs to allow proper alignment of the great vessels over their respective ventricles. The signals that initiate apoptosis in this setting are unknown. The aim of this study was to characterize the cells undergoing apoptosis in the cardiac OFT myocardium and the cells that may influence this process. Two cell populations that may initiate apoptosis of the cardiomyocytes are the cardiac neural crest (CNC) cells and epicardial cells. We examined stage 30–31 chick embryos that had undergone removal of the CNC cells or had delayed epicardial growth for alterations of apoptosis. Removal of the CNC cells did not reduce the levels or pattern of apoptosis in the OFT myocardium. In contrast, impeding the growth of the epicardium over the OFT resulted in a 57% reduction in apoptotic cells in the OFT myocardium. Analysis of the apoptotic cells within the OFT myocardium showed that as many as 92% of them expressed cardiomyocyte markers. In the quail, the endothelial marker QH1 identified a component from the epicardium, endothelial cells, in regions where apoptosis is elevated in the OFT myocardium. These results suggest that a component from the epicardium, possibly endothelial cells, is required for the initiation of apoptosis in OFT cardiomyocytes.

Conditioning Effect of Blood Flow on Resistance Artery Smooth Muscle Myosin Phosphatase

Myosin phosphatase is the primary effector of smooth muscle relaxation and a target of signaling pathways that regulate vascular tone. The mesenteric small resistance artery and large vessel smooth muscle express distinct isoforms of the myosin phosphatase targeting subunit (MYPT1), and the isoforms in the small resistance artery switch in a disease model of altered blood flow. We thus hypothesized that small resistance artery smooth muscle phenotype is responsive to altered blood flow. To test this hypothesis alternating pairs of rat second order mesenteric arteries were ligated so that the upstream first order mesenteric artery (MA1) is under chronic low flow and the adjacent first order mesenteric artery under chronic high flow. The initial response was similar in high flow and low flow MA1, and included rapid reduction in MYPT1 and switch to the 3′ alternative exon skipped/leucine zipper positive MYPT1 isoform. Between 14 to 28 days, MYPT1 abundance was restored along with reversion to the MYPT1 leucine zipper− isoform under chronic high flow. In contrast, under continued low flow, there was further switching to the MYPT1 leucine zipper+ isoform. As would be predicted based on the switch to the MYPT1 leucine zipper+ isoform, the sensitivity for relaxation to the NO donor SIN-1 and to cGMP was increased in the Day28 low flow first order mesenteric artery. We conclude that pulsatile blood flow conditions the phasic program of gene expression in the small resistance artery smooth muscle. The loss of this conditioning effect significantly increases the sensitivity to vasodilator signals in the setting of chronically reduced blood flow.

Endothelin-1 Alters the Contractile Phenotype of Cultured Embryonic Smooth Muscle Cells

Smooth muscle tissues may be classified into phasic (fast) or tonic (slow) contractile phenotypes. This study was initiated to examine the specification of these phenotypes during development and the role of growth factors in this process. We used myosin light chain 17 (MLC17) and myosin heavy chain transcript splice variants as markers of the tonic (aortic) and phasic (intestinal) smooth muscle phenotypes in chick embryos. By reverse transcription-polymerase chain reaction, we determined embryonic days 6 to 16 to be a critical period for the establishment of these phenotypes. During this period, endothelin-1 is present at 40-fold-higher levels in aortic compared with intestinal tissues. To test the hypothesis that endothelin-1 may be involved in establishing the aortic (tonic) phenotype, we developed a system in which embryonic smooth muscle cells exhibit phasic and tonic contractile properties in vitro. Single-cell force measurements showed that cultured embryonic gizzard (phasic) cells developed force more rapidly (8 +/- 2 seconds) and achieved greater force (3.0 +/- 0.7 microN) than did cultured embryonic aortic (tonic) cells (20 +/- 0.7 seconds, 0.76 +/- 0.01 microN; P < .05) in response to depolarization. Chronic exposure of the phasic (gizzard) cells to endothelin-1 prolonged the time to peak force (24 +/- 3 seconds) and reduced the peak force (1.0 +/- 0.1 microN), so that the contraction resembled the tonic type. This effect, mediated by the endothelin-A receptor, was associated with a shift in MLC17 splicing to the tonic pattern. These results demonstrate that endothelin-1 is highly enriched in developing aortic compared with intestinal tissues and can convert phasic smooth muscle cells to the tonic type in vitro, suggesting a role for this growth factor during development in determining the contractile phenotype of smooth muscle cells.

Dynamic patterns of apoptosis in the developing chicken heart

The outflow tract (OFT) is abnormal in many congenital heart defects. One critical mechanism for morphogenesis of this complex structure is apoptosis. Chicken embryos (stages 19–38; ED4–10) stained with a fluorescent supravital lysosomal dye (LysoTracker Red; LTR) revealed the three‐dimensional relationship between structural changes and apoptosis. The LTR staining peaked in the OFT myocardium at stages 27–32, consistent with our previous analyses using other apoptosis assays. While LTR stained under both the pulmonary artery and the aorta, it was most prevalent in the subaortic myocardium before its elimination. Furthermore, LTR staining was most abundant in the myocardium under intensely cytokeratin‐positive, thick epicardium. These data support the hypothesis that temporally and spatially restricted apoptosis in the OFT myocardium allows the aorta and pulmonary artery to dock at the appropriate angle and level with the proper ventricle. These data also support a relationship between the differentiating epicardium and cardiomyocyte apoptosis. Developmental Dynamics 229:489–499, 2004.

Altered hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies.

The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5‐positive tissues were also specifically positive for nuclear‐localized hypoxia inducible factor‐1 alpha (HIF‐1α), the oxygen‐sensitive component of the hypoxia inducible factor‐1 (HIF‐1) heterodimer. This led to our hypothesis that there is a “template” of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study, we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75–40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF‐1α protein expression, and its downstream target genes without high mortality. We also altered HIF‐1α gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF‐1α (AdCA5). We assayed for coronary anomalies using anti‐alpha‐smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF‐1 regulation of gene expression. Developmental Dynamics 238:2688–2700, 2009.