Mechanical activation of signaling pathways in the cardiovascular system

ELSEVIER

BRIEF REVIEWS pose the system. Likewise, these physical forces also influence the phenotype of these tissues upon which they impact, resulting in chronic alterations (both adaptive and pathological) in the phenotypes of these cells and tissues. To influence the biology of cells, these stimuli, which exist in the physical domain, must be converted into signals in the biochemical language of the cells. This process has been referred to as mechano-chemical signal transduction, or mechanotransduction. The actual nature of these conversion events is very poorly understood, with little being known in many cases regarding the actual signals that initiate the cascade of events leading to altered phenotype or behavior. This review attempts to distinguish between players definitively identified as participating in the process of mechano-chemical signal transduction in the cardiovascular system, and those whose actions have been inferred through correlation or concurrence.

Mechanical Activation of Signaling Pathways in the Cardiovascular System Peter A. Watson

Mechanical forces are constantly exerting stress upon the tissues and cells of the cardiovascular system. To influence the biology of cells, these stimuli, which exist in the physical domain, must be converted into signals in the biochemical language of the cells. This process has been referred to as mechano-chemical signal transduction, or mechanotransduction. Although a great deal is known about which aspects of cardiovascular biology are influenced or dictated by physical forces, a great deal of uncertainty exists about which of the many signaling pathways that respond in cardiovascular cells to mechanical stimuli specifically regulate mechanosensitive aspects of the “cardiovascular phenotype.” Even less is known regarding the identity and function of structures and catalysts that operate at the physical-biochemical interface and act to convert physical energy into signals of biological relevance. This article presents what is known regarding signaling pathways in cells of the cardiovascular system, which have been shown empirically to respond to mechanical stimuli, and what can be inferred The Nature of Physical Forces in from biochemical and pharmacological studies in cultured cardiovasthe Cardiovascular System and the Tissues and Cells That Sense cular cells regarding the potential for certain signaling pathways to be and Respond to These Forces involved in the manifestation of mechanically responsive phenotypes in The physical forces to which cells in the the cardiovascular system. (Trends Cardiovasc Med 1996;6:73-79). l

Mechanical forces are constantly exerting stress upon the tissues and cells of the cardiovascular system. The cardiovascular system is a closed circuit, which is responsible for containing and transporting the serum and corpuscular components that make up blood. The actions of gravity upon the fluid within the vasculature, as well as flow of blood down the pressure gradient established

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and maintained within the system by the pulsatile ejection of blood from the heart, all impart distention or shear forces upon the tissues of the cardiovascular system. The flow of blood, the pressure of the fluid within the system, and the filling and contractile pressures that alternately distend and compress the chambers of the heart, are modulated as a function of these physical parameters. Thus, these forces are experienced and sensed by both specialized neurological structures within the system, as well as by all the cells that comScience

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cardiovascular system are exposed are limited to three general categories: shear, stretch, and vibration. The type of forces a cell type experiences is dependent upon its location in the cardiovascular system. As shear forces are generated as the result of direct exposure to the circulating blood and its flow, both turbulent and laminar, only those cells that actually line the structures of the vasculature and the heart experience these physical forces. The cells that are the principal sensors for such stimuli in the cardiovascdar system are the vascular endothelial cells, which are capable of responding to various stimuli with a wide variety of endocrine functions that

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influence the behavior of other cells in the vasculature. A much wider spectrum of cell types is exposed to the latter two types of stimuli, stretch and vibration. Flow and pressure in the vessels of the vasculature generate forces tangential to the direction of flow within the vessel. These forces distend the elastic walls of these vessels, resulting in the stretch of cells in all layers of the vessel. The cell types in the vessel walls that are exposed to these forces include the endothelial cells, vascular smooth muscle cells, and fibroblastic cells. The distensional forces resulting from pressure in the chambers of the heart lead to expansion or stretch of the various layers of myocytes in the myocardium. In addition, the rhythmic contraction of the myocytes in the heart also generates distensional forces that are experienced by all cells in the chamber walls. All the cardiovascular cell types just described demonstrate some behavioral or adaptational change in phenotype in response to mechanical forces. These findings have been derived from experiments performed both in vivo as well as in isolated cells in culture or isolated tissues in vitro. The circumstance of the vessel wall is unique in that both shear forces and stretch forces can be imposed simultaneously upon the endothelial cells that line them. These forces can generate, depending upon the parameter examined, either similar or opposing effects on endothelial cell phenotype.

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Signaling Events Demonstrated Through Direct Measurement to Be Activated in Cardiovascular Cells in Response to Mechanical Stimuli

Cardiac Cells Contractile activity and stretch have been used as mechanical stimuli to induce cardiac myocyte growth and adaptation. A very limited number of signaling pathways have been implied to be activated directly by stretch, deformation, or contraction of cardiac myocytes or any cell type in the ventricular myocardium. Indeed, examination of the literature might lead one to conclude that only one signaling pathway may, at this point in time, be ascribed true mechan74

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otransducing behavior, stretch-activated is unknown, however, whether phospholiion channels (Sachs 1987). Studies im- pase C is the initial site of action where ply that stretch-activated ion channels, conversion from physical force to biowhich can be activated by mechanical chemical signal occurs, or whether activaevents, are present in the membranes of tion of phospholipase C falls downstream isolated chick cardiac myocytes (Sigurdin a signaling cascade initiated by another son et al. 1992). Deformation with an mechanotmnsducer. inert rod of isolated chick cardiac myoExperiments performed in densely cytes previously loaded with calciumplated, spontaneously and synchrosensitive dyes stimulates the focal influx nously contracting neonatal rat cardiac of calcium into these cells (Sigurdson et myocytes have demonstrated that conal. 1992). This calcium influx is followed tractile activity induces increased rates by a generalized and directional increase of protein synthesis (McDermott and in intracellular calcium throughout the Morgan 1989) and ribosome biogenesis myocyte (Sigurdson et al. 1992). That (McDermott et al. 1991, McDermott and myocytes demonstrate increased cal- Morgan 1989), as well as selective excium influx in a focal manner dependent pression of a number of structural genes upon the deformation in an adjacent (P. Watson unpublished observations). myocyte suggests that mechanical couSuch contractile activity has been shown pling, and not electrical coupling, ac- to result in translocation of protein kicounts for the calcium influx generated nase C activity from the cytoplasm to the in these myocytes (Sigurdson et al. sarcolemma (Al10 et al. 1992), a marker 1992). Patch-clamp analysis of the memfor protein kinase C activation. In carbranes of these myocytes indicated the diac myocytes, protein kinase C has presence of nonselective cation stretchbeen implicated as a signal coupled to activated channels whose activity could anabolic responses with a number of be blocked by addition of 20 pM gadostimuli, including alpha-adrenergic agolinium to the patch medium (Sigurdson nists (Allo et al. 1992) and endothelin et al. 1992). Changes in intracellular flu(Neyses et al. 1991). These observations orescence stimulated by mechanical suggest that activation of protein kinase prodding of myocytes in culture were C may link stimuli to anabolic responses also blocked by gadolinium, implying associated with cardiac myocyte growth. that direct activation of stretch-activated Mitogen-activated protein (MAP) kichannels was responsible for the nase activity has been linked to a numchanges in intracellular calcium (Sigber of aspects of hypertrophic phenourdson et al. 1992). However, a role for types in cardiac myocytes. Two studies stretch-activated channels in altering have reported that cardiac cells can recardiac myocyte phenotype or behavior spond to mechanical forces with inhas not been defined. creases in MAP kinase activity. InA number of systems that respond to creased coronary perfusion pressure mechanical challenge have been shown to (120 mm Hg; 5 min) results in a 2- to manifest me&no-sensitive activation of phospholipase C, which generates two in- S-fold increase in MAP kinase and MEK tracellular signaling molecules as the re- (MAP kinase kinase) activities in isolated perfused rat heart (Lazou et al. sult of hydrolysis of membrane inositol phospholipids. These signals, inositol tris- 1994). Additionally, a modest activation phosphate (IP,) and 1,2 diacylglycerol of MAP kinase activity by mechanical (DAG), am responsible for stimulating the forces was demonstrated in isolated cardiac myocytes subjected to passive release of specific pools of intracellular calcium and activation of protein kinase C stretch (Yazaki et al. 1993, Takewaki et al. 1995). MAP kinase activation has activity, respectively. Stretch of cultured neonatal rat cardiac myocytes has also been linked to a cascade of events that been shown to induce growth (Sadoshima leads to expression of the immediate et al. 1992 and 1993, Kumoro et al. 1991). early gene c-fos in cardiac myocytes in Stretch has also been shown to elicit acti- response to angiotensin II (AD) (Savation of the phospholipase C-dependent doshima et al. 1995). The potential involvement of AD in stretch-induced ansignaling cascade in cultured neonatal cardiac myocytes, as evidenced by the abolic responses in cultured cardiac generation of inositol trisphosphate and myocytes is somewhat controversial, tetrakisphosphate (Dassouh et al. 1993). It and is discussed later in this article.

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Recent studies have suggested that activation of the Na+/I-I+ antiporter may be a primary site of action for stretch in cultured cardiac myocytes (Takewaki et al. 1995) and may influence the anabolic state of the ventricular myocardium in response to pressure overload (Sugden and Fuller 1991, Gaitanaki et al. 1990). Experiments performed on isolated neonatal rat cardiac myocytes, cultured on elastic membranes, indicate that stretchactivated increases in MAP kinase activity and protein synthetic rates can be modestly, but significantly, attenuated by treatment of cells with the Na+/I-I+ antiporter-antagonist Hoe 694 (Takewaki et al. 1995). Although indicative of some effect of stretch upon the Na+/H+ antiporter, these results are not compelling with regard to the potential importance of this particular signaling pathway in the regulation of stretch-induced cardiac phenotype and behavior. Increased aortic perfusion pressure resulted in a rapid increase in CAMP and the activity of the &IMP-dependent protein kinase (PKA) in the isolated perfused rat heart (Watson et al. 1989). This increase in CAMP and PKA in perfused hearts was coupled to increases in rates of both protein synthesis and ribosome biogenesis (Watson et al. 1989). Pressure-induced increases in CAMP in the heart, and the ability of increases in CAMP to alter cardiac growth are controversial. Similar, but not identical, experiments performed in the perfused heart model failed to demonstrate increases in CAMP content concomitant with pressure-induced increases in protein synthesis (Bogoyevitch et al. 1993). Additionally, no effects of elevated CAMP were found on anabolic processes in neonatal rat cardiac myocytes in culture (Sadoshima and Izumo 1993a, Simpson et al. 1986). However, increased CAMP in adult feline cardiac myocytes in culture resulted in accelerated rates of protein synthesis (Decker et al. 1995). This disparity points out one of the difficulties encountered in examining the results of experiments performed in vitro utilizing a broad spectrum of model systems, and subsequently drawing conclusions regarding effects observed in vivo. Additional levels of complexity regarding the potential for crosstalk between signaling pathways also make interpretation of experimental results across model systems difficult. For example, activation of protein kinase C has been shown to increase significantly TCM Vol. 6, No. 3, 1996

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synthesis of CAMP by type V (Kawabe et al. 1994) and type VII (Watson et al. 1994) adenylyl cyclases, both of which am expressed in the heart (Krupinski et al. 1992).

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Vascular

Cells

As just discussed, the location and function of cells in the peripheral vasculature dictates that a somewhat broader spectrum of mechanical challenges are experienced by these cells. As was the situation with cells in the heart, a limited number of signaling pathways have been demonstrated through direct measurement to be activated in vascular cells by mechanical challenge. The activity of nonselective cation stretch-activated channels has been detected in the membranes of cultured aortic endothelial cells by patch-clamp techniques (Lansman et al. 1987). The possible involvement of stretch-activated channels in regulatory events in cultured endothelial cells in response to shear stress has also been implied (Lansman et al. 1987, Nat-use and Sokabe 1993). Mechanical challenge of cultured aortic endothelial cells by shear stress as well as cyclic stretch has been shown to increase protein kinase C activity and IP, genemtion (Nollert et al. 1990, BhagyaUshmi et al. 1992, Rosales and Sumpio 1992, Shen et al. 1992, Dassouli et al. 1993). The responses in phospholipase C-generated signals to mechanical challenge in these cells appear to be related more closely to the alteration of the intensity of strain than to the new level of strain that was imposed upon the cells (Rosales and Sumpio 1992, Brophy et al. 1993, Winston et al. 1993). Thus, an increase or decrease in the strain load on these cells stimulates an acute increase in IP, concentration, which sub sequently accommodates-following prolonged exposure to the altered level of strain-decaying to steady state levels ap proaching values found in cells prior to the alteration in strain (Rosales and Sumpio 1992). Limited information exists regarding the subsequent effects of these mechanically-induced signals on endothelial cell biology or phenotype, although activation of c-fos expression in cultured aortic endothelial cells appears to fall downstream of mechanically induced phospholipase C activation (Ranjan and Diamond 1993, Yazaki et al. 1993). Experiments performed in isolated cerebral arterioles indicate that myogenic ScienceInc., 1050-1738/96/$15.00

responses of these vessels to increased flow are apparently protein kinase Cdependent events. Experiments performed in vessels both containing endothelial cells (0~01 et al. 1991) and denuded of endothelial cells (G. 0~01 personal communication) demonstrate myogenic responses in response to flow. The response in intact vessels is apparently distal to an increase in phospholipase C activity, potentially activated by a G protein-dependent mechanism (0~01 et al. 1993). This observation implies that vascular smooth muscle cells, in addition to vascular endothelial cells, sense and respond to flow-generated vessel expansion with the initiation of a signaling cascade that ultimately results in increased protein kinase C activity.

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The “Chicken-and-Egg” Conundrum of Signal Transduction Pathway Activation, Signal Crosstalk, and Cellular Heterogeneity

A wide variety of models and stimuli have been used to elicit alterations in the phenotypes of cardiovascular cells. Attempts have been made to draw conclusions regarding similarities in the responses elicited in these cells by humoral agents and mechanical stimuli, and subsequently to draw conclusions from these cortelations regarding the involvement of specific signaling pathways in the regulation of these mechanically induced events. In most circumstances, data are lacking that truly link a specific signaling pathway both to mechanical stimuli and to a mechanically induced change in cardiovascular cell phenotype. The studies that do exist regarding correlations between signaling events and alterations in cell structure and function must be carefully interpreted with regard both to the great degree of signal crosstalk that occurs within cells, and to cellular heterogeneity present in the model systems used for such studies. Cardiac

Cells

A significant number of peptide hormones and cytokines that induce hypertrophic growth in cultured neonatal rat cardiac myocytes have also been shown to be synthesized and released from cardiac myocytes. Release of certain of these agents from cardiac myocytes following mechanical challenge has been demonstrated. Specific interest has fo-

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cused upon the angiotensin II (AH). A number of specific observations in cultures of cardiocytes have led to this interest. The presence of mRNAs encoding angiotensinogen (Sadoshima and Izumo 1993, Dostal et al. 1992) and renin has been demonstrated in cardiocytes (Dostal et al. 1992). Release of AI1 from stretched cardiocytes has been documented (Sadoshima et al. 1993), and the ability of AI1 to induce certain aspects of hypertrophic phenotypes in cultured cardiocytes, coupled with the ability of AT,-angiotensin II receptor antagonists to block stretch induction of these hypertrophic responses (Sadoshima et al. 1993), argues strongly for a role of AI1 in certain aspects of mechanically induced growth. However, drawing definitive conclusions from the observations that (a) AI1 is released from stretched cardiac myocytes and (b) AI1 can elicit aspects of the stretch-induced hypertrophic phenotype in these cells is somewhat dangerous. Results from studies regarding treatment of cultured cardiac myocytes with alpha-adrenergic agonists and subsequent activation of MAP kinase activity indicate that a simple linear pathway of signal transduction cannot account for all aspects of hypertrophic phenotypes elicited in cardiac myocytes (Thorburn et al. 1994). Although changes in immediate-early gene expression are prevented by purine nucleotides that antagonize MAP kinase activation, alterations in cytoskeletal structure that also occur in cardiac myocytes during alphaadrenergically stimulated hypertrophy are insensitive to MAP kinase inhibition (Thorbum et al. 1994). As certain aspects of the AH-induced hypertrophic response have been ascribed to activation of MAP kinase activity (Sadoshima et al. 1995) this result implies that other signaling pathways must be involved in the manifestation of stretch-induced growth in cardiac myocytes. Problems also have arisen regarding whether AI1 acts directly to elicit these effects in cardiac myocytes. The ability of AH to elicit general growth responses in purified cultures of neonatal rat cardiac myocytes is not a consistent finding. In experiments performed in my laboratory, treatment of purified cultures of neonatal rat cardiac myocytes with AI1 at doses that have previously been shown to stimulate hypertrophic growth and maximal c-fos mRNA accu76

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Cardiomyocyte

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Figure 1. Angiotensin II fails to stimulate the accumulation of either total cellular protein or mechanosensitive cytoske1eta.l protein mRNAs in purified cultures of neonatal rat cardiac myocytes. Upper pan& Accumulation of cellular protein per cardiac myocyte cell (corrected for cellukr DNA in both KCl-arrested and contracting cardiac myocyte cultures in response to norepinephrine + propranolol(1 Om6 M and 2 x 10e5 M), respectively, or angiotensin II after 72 h of treatment. (*) Denotes statistical significance (p < 0.05 relative to arrested control cells, and (#) denotes statistical significance ( p < 0.05 relative to contracting control cells. Lower panel: Content of specific cytoskeletal protein mRNAs, as determined by Northern blot analysis, in KC1arrested, arrested and treated with phenylephtine ( 10m4 M), isoproterenol(1 Oe6 M), or angiotensin II ( 10m6 M), or contracting cultures of neonatal rat cardiac myocytes.

mulation in these cells (lop6 M) (Sadoshima et al. 1993) fails to cause a significant increase in cell protein content or accumulation of contraction or stretch-sensitive cytoskeletal protein mRNAs (Figure 1) (Watson et al. 1995).

An alternative explanation is that AI1 acts to alter the production and release of other agents from both cardiac myocytes and nonmyocytes, such as endothelin (Shubeita et al, 1990) or transforming growth factor-beta (TGF-S) (Omura et al. 1995, Sadoshima and Izumo 1993b, Everett et al. 1994), which can also elicit growth responses in cardiac myocytes (Ito et al. 1991, Neyses et al. 1991). A specific question that is critical to this discussion is “what signaling mechanism is responsible for stretch-induced AI1 re-

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lease from cardiac myocytes?” No convincing data exist as to what signals might be generated in direct response to mechanical stimulation, which might account for stretch-induced AII release from cardiac myocytes. It could be speculated that increases in intracellular calcium, demonstrated to occur in cardiac myocytes subjected to mechanical deformation (Sigmdson et al. 1992) could be involved in the release of AI1 and other agents from mechanically challenged cardiocytes. Increases in intracellular calcium have been shown in a variety of cell types to be involved in endocytic vesicle fusion and release of a number of peptides and cytokines. This author is unaware of specific data supporting or refuting this speculation in the literature. Recent work by Kaye et al. (in press) implies that mechanical forces such as those that accompany contractile activity can lead to increased membrane permeability and the release of peptides and cytokines [such as basic fibtoblast growth factor (bFGF)] from cardiocytes through nontraditional mechanisms. Vascular

Cells

The endothelial cells that line the vasculature serve both as mechanosensors and “master regulators” of the reactive and adaptive responses of the vasculature to increased flow and pressure. A wide variety of vasoactive compounds are synthesized and released by vascular endothelial cells. Production and release of several of these agents, as well as the content of mRNAs encoding some of these agents, are influenced in endothelial cells by flow and stretch. These include endothelin-1 (Morita et al. 1995, Malek et al. 1993), tissue plasminogen activator (Diamond et al. 1990, Iba et al. 1991), bFGF (Malek and Izumo 1992), platelet-derived growth factor (PDGF) (Hsieh et al. 1992) and nitric oxide (NO) (Nor-is et al. 1995, Korenaga et al. 1994, Dainty et al. 1990). In most cases, the actual signaling cascade initiating the responses in these vasoactive compounds in endothelial cells is unknown. In the specific case of acute increases in NO production and release, both extracellular calcium and ATP appear to be involved in the initiation of this process (Korenaga et al. 1994). Similar to the responses seen in signal generation in the cardiac myocyte, step changes in flow produce increases in NO synthesis, which are independent from the new steady state intensity of

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Acknowledgments

The author thanks Dr. Ross Hannan for constructive conversation and input into the preparation of this manuscript.

A

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Kinase C

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2. Signal transduction pathways and potential crosstalk regulation vascular cells by mechanical forces. A number of signal transduction cardiac or vascular cells have been demonstrated to be sensitive to changes environment. Signaling pathways whose activity is modulated concomitant stimulation of cardiovascular cells are shown in bold italics, with potential signaling pathways and downstream effects also presented.

shear, exceeding NO synthesis rates related to the intensity of steady state laminar flow (Noris et al. 1995).

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Conclusions

Although a great deal is known about which aspects of cardiovascular biology are influenced or dictated by physical forces, a great deal of uncertainty exists about which of the many signaling pathways that respond in cardiovascular cells to mechanical stimuli (Figure 2) specifically regulate mechanosensitive aspects of the “cardiovascular phenotype.” Even less is known regarding the identity and function of structures and catalysts that operate at the physicalbiochemical interface and act to convert physical energy into signals of biological relevance. Important questions remain to be answered regarding why certain mechanical challenges (and the signals they generate) elicit adaptive responses that improve function of cardiovascular tissues (volume overload in the heart), whereas others elicit degenerative pathologies (pressure overload/cardiac failure). Much more will need to be discovered about this energy conversion

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activated in cardiopathways in either in their mechanical with mechanical crosstalk between

process before more effective interventions into mechanically related pathologies in the cardiovascular system can be designed. Most of the physiological and pathological phenotypes elicited by mechanical forces in vivo are the net result of subtle changes in the physical environment and biochemical pathways, which, with the passage of time, manifest as longterm changes of significant proportions. Thus, the in vitro model systems that are used in studies of these mechano-chemical signaling events tend to exaggerate the degree of the stimulus in order to maximize the changes in these systems (arrest versus contraction in cardiac myocytes, for example). In accepting these limitations, it will be necessary to correlate all conclusions drawn from in vitro model systems to changes observed in the intact heart and circulatory system. Investigations utilizing molecular analysis of the regulatory regions of genes that encode proteins that comprise aspects of adaptive and pathological phenotypes in cells of the cardiovascular system (Resnick et al. 1993, Aoyagi and Izumo 1993) offer significant hope with regard to sorting the true signals elicited in these cells by mechanical stimuli from the noise of crosstalk.

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form of mammalian adenylyl cyclase expressed widely in mouse tissues and in S49 mouse lymphoma cells. J Biol Chem 269: 28,893-28,898. Watson PA, Hannan R, Carl LL, Giger KE: 1995. Desmin gene expression in cardiac myocytes is responsive to contractile activity and stretch. Am J Physiol (in press). Winston F, Thibault L, Macarak E: 1993. An analysis of the time-dependent changes in

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Retroviral Vectors to Study Cardiovascular Development Takashi Mikawa, Jeannette Hyer, Naoki Itoh, and Yan Wei The functioning of the vertebrate heart depends upon the proper organization of its complex structure: atria1 and ventricular myocytes, the conduction system, the fibrous skeleton that forms the chambers and valves of the heart, the coronary vessels, and neural elements. Cell-type specific transgenic models for addressing molecular mechanisms regulating heart development necessitate controllable gene insertions into the precursors of each cell type forming the heart. The unique properties of retrovirus-based shuttle vectors provide a powerful tool for (a) identifying the origin and lineage relationships of all cell types forming the heart: and (b) gene targeting to the selected cell type and time point during heart development. (Trends Cardiovasc Med 1996;6:79-86).

bers into each transfected or infected cell. Furthermore, the introduced genes may not be stable and are often lost. These procedures may be appropriate for experiments based on tmnsient expression, but may not be optimal for gene analyses over the entire course of development. Tmnsgenie mouse systems have the advantage that all cells in the animal possess the same gene copy number, generally in a stable integrant. However, it is currently difficult, if not impossible, to target a selected gene in each cell type of the heart at a defined time point during development with a transgenic mouse. In addition, because the cardiovascular system is the first organ system to develop and function in the embryo, gene knockouts that result in the ablation or alteration of crucial factor(s) necessary for heart development

will give rise to embryonic lethals (Henkemeyer et al. 1995) or to secondary effects of impaired circulation or cardiac function, which complicate the mechanistic interpretations of the mutations. Some of these problems can be overcome by utilizing a replication-defective retrovirus. A retrovirus system permits the insertion and stable integration of single copy transgenes, and the targeted insertion of the transgenes into defined cell types by direct

ation

The heart of higher vertebrates including birds and mammals is established through the integrated and sequential processes of cell commitment, morphogenesis, cell proliferation, and cell movements. To date, little is known about molecular mechanisms regulating these develop mental processes of the heart, largely owing to the lack of a simple protocol that satisfies certain requirements for successful genetic manipulation within the developing heart, namely, reproducibility and high levels of expression of the exogenous gene: maintenance of embryonic viability; and regulated expression of trarzs-genes

Takashi Mikawa, Jeannette Hyer, Naoki Itoh, and Yan Wei are at the Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021, USA.

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only in the selected cell types and at defined time points. Several molecular ap proaches have been taken to express exogenous genes in the heart. These include (a) transgenic mice (Steinhelper et al. 1990, Lee et al. 1994, Henkemeyer et al. 1995) [for a review, see Robbins et al. (19931; (b) direct injection of expression vector DNAs (Leinwand and Leiden 1991); (c) DNA-vital mediated gene transfer (Stratford-Penicaudet et al. 1992); and (d) retmviral-mediated gene transfer (Mikawa et al. 1992a and b, Mikawa and Fischman 1992). None of these procedures satisfy all three requirements (Table 1). For example, transfection methods and DNA-viral transmission systems permit gene delivery into both dividing and nondividing cells such as cardiomyocytes in adult hearts. However, these two procedures do not permit transfer of defined gene copy num-

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microinjection.

Addi-

tionally, one can achieve high-level expression of transgenes without tissuespecific promoter-enhancer elements in the targeted cell types. The resulting animal has the advantage that any alterallows

the generation

of somatic

(heart) mosaics consisting of cells expressing transgenes and those undergoing normal development. As a result of this latter point, if the transduced cells are restricted to minimal areas of the heart, the mosaic organ may not give rise to embryonic lethals, but instead may permit the analysis of phenotype of the targeted cells.

l

Life Cycle of Fktroviruses

The retroviral particles of -100 nm in diameter consist of two major subcomponents: the central core, which contains two copies of a single-stranded RNA genome and reverse transcriptase molecules with nucleoproteins, and the membrane coat bearing specific viral envelope glycoproteins. The infectious virions start their life cycle (Figure 1) by binding to the surface

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