SPECIAL TOPIC
Prologue: low-molecular-weight GTPases in the heart and circulation

¹ Weis Center for Research, Pennsylvania State University College of Medicine, Danville, Pennsylvania 17822; and ² Rammelkamp Center for Education and Research, MetroHealth Medical Center and Case Western Reserve, Cleveland, Ohio 44109

	ARTICLE

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This current Special Topic section of the AJP: Heart & Circulatory Physiology focuses on five studies that examine mechanisms involving small GTPase-binding proteins (3, 9, 11, 16, 20). Although formerly primarily in the domain of cancer research, it has become increasingly clear that these proteins will be important in transducing the molecular changes in cardiovascular diseases as well. Studies of the role of small GTPases in the heart, vascular smooth muscle, and endothelial cells have focused on functions related to the regulation of gene expression, cell growth, movement, development, and cytoskeletal organization. The goal of this prologue is to put these molecules into perspective for the readers of AJP: Heart & Circulatory Physiology, in view of the fact that these proteins will be appearing in increasing frequency in the cardiovascular physiology literature.

The Ras-related or small GTPases comprise a superfamily of genes encoding small GTP-binding proteins (20-25 kDa). These GTPase molecules play an important role in integrating signals from cell surface receptors to intracellular responses. The low-molecular-weight GTPases must be distinguished from the more generally recognized heterotrimeric regulatory G proteins, which are two to three times larger than the monomeric small GTPase proteins with more rapid GTPase activity. The large heterotrimeric G proteins are membrane bound and regulate the downstream effector response to membrane-bound, receptor-mediated activity, whereas, the small GTPases are located throughout the cell, depending on their function.

On the basis of structure and function, six subfamilies of low-molecular-weight GTPase proteins have been identified, which include Ras, Rho, Rab, Arf, Sar, and Ran. The original meaning of the abbreviations for small GTPases in many cases has been forgotten because the origin of the terms is based on the oncogenes originally identified. Since then, the study of these proteins has expanded to many different tissues, and only the cryptic acronyms remain. Nonetheless, the derivation of the acronyms, although not always critical to their function, may be of interest. They include Ras (t arcoma virus, which is involved in cell growth and differentiation), Rho (as mologous), Rab/Ypt (yeast protein wo, which is involved in regulating exocytosis and endocytosis), Arf (DP-ibosylation actor, which is involved in vesicle formation and budding), Sar (ecretion-ssociated and as superfamily-related gene), Ran (s-related A transport, which is involved in the transport of RNA and proteins across the nuclear membrane), and Rap (s-related protein, which is located in granules of the Golgi and endoplasmic reticulum and involved in growth and development). Closely related to Ras are the Ral-A and Ral-B proteins (s ated, which appear to regulate the activity of exocytic and endocytic vesicles).

Clearly more molecules will be described shortly in this rapidly expanding field. These Ras-related proteins have similar sequences and are molecular switches that trigger many cellular functions. For example, the Rho subfamily of Ras [Rho A, B, C, D, E/Rnd3, Rnd1/Rho6, Rnd2/Rho7, RhoG, Rac1, 2, 3 (s-related 3-botulinum toxin substrate), Cdc42 (homologous to yeast ell ivision ycle gene ), TC10, and TTF] link cell surface receptors to the organization of the actin cytoskeleton and assembly of focal adhesions and actin stress fibers (for review see Ref. 10). As noted above, in the late 1970s and early 1980s, the initial work on small GTPases focused on viral oncogenes. The location of Ras was identified on the inner face of the plasma membrane. Ras proteins bound GDP and GTP with high affinity, and it was shown that the Ras proteins also had an intrinsic GTPase activity (5).

The most logical, but not the only, link to cardiovascular disease for these proteins are the states of hypertrophy and hyperplasia, which have many features in common with neoplasia, where the original work was done. Indeed, it has been shown that the normal and pathological myocyte response of cardiac hypertrophy is regulated partly by the members of the small GTPase family. Transgenic mice that express oncogenic Ras in the ventricle have morphological, physiological, and genetic markers of severe hypertrophy (7). An in vitro model of ventricular hypertrophy, the ₁-adrenergic-induced growth in neonatal ventricular cardiac myocyte culture, has been used to investigate the role of small GTPases in the hypertrophic response, including changes in expression of contractile protein genes, myofibrillogensis, and atrial natriuretic factor (ANF) gene expression. The details of mechanisms by which ₁-adrenergic agonists stimulate transcriptional activation of the ANF gene and the myofibrillar organization in this model are being delineated by several groups (for review see Ref. 4). Thus far both the Rho and Ras family GTPases have been clearly demonstrated to play a role in pathways in this in vitro model with evidence of cross talk between pathways. Ras via mitogen-activated protein kinase kinase (MEKK) and c-Jun NH₂-terminal kinase (JNK) was shown to stimulate ANF gene expression (13). However, other studies suggested that this was not the only pathway. A parallel path via Rho was implicated in the production of ANF in the hypertrophic response (14). Involvement of Rho in the cytoskeletal function of cardiac myocytes has been demonstrated by several groups. Rho A in neonatal rat cardiomyocytes has been shown to be activated by another hypertrophic stimulus, angiotensin II, and mediates the angiotensin II-induced formation of premyofibrils (2). Also, the role of Rho in cardiomyocytes evaluated using botulinum toxin C3, an inhibitor of Rho GTPase, and expression of activated and dominant negative forms of the protein show that Rho and its downstream effector, Rho kinase, have an important role in the structural changes in hypertrophy (6). However, there is also a contrasting report that Rho regulates gene expression but not actin organization (19).

Studies using adenoviral-mediated gene transfer of constitutively active and dominant negative isoforms of Rac1 (member of Rho subfamily) to assess the role of this small GTPase in cardiac myocytes suggest that Rac1 is also an essential element of the signaling pathway leading to hypertrophy. Expression of the constitutively active isoform of Rac1 in neonatal cardiac myocytes results in increase in actin filaments, sarcomeric organization, and an increase in cell size like that of ligand-stimulated hypertrophy. In contrast, expression of the dominant negative Rac1 reduced the morphological changes associated with phenylephrine stimulation (12). Rho was also shown to have an important role in another in vitro model of hypertrophy induced by mechanical stress. Stretch induced the activation of extracellular signal-related kinases (ERKs), expression of skeletal -actin and c-fos genes, and an increase in protein synthesis in neonatal cardiac myocytes (1). Recently, Sah et al. (15) showed that overexpression of RhoA in an in vivo model leads to sinoatrial (SA) and atrioventricular (AV) nodal changes that produce atrial fibrillation and AV nodal block as well as development of ventricular failure. They did not, however, find myocardial hypertrophy, which certainly provides impetus for further studies in this arena.

Much of the recent work on small GTPases that has appeared in the cardiovascular literature has centered on the actions of the Rho GTPases. Therefore, it is not surprising that all five of the articles in this series deal with the actions of members of this family in heart and vasculature. In one of the studies in this issue, Morissette et al. (9) continue their work on defining the Rho pathway described above. This study further delineates the contributions of the Rho pathway with the demonstration that a protein kinase C-related kinase is a downstream effector of Rho in the ANF transcription component of the response to -adrenergic stimulation.

The Rho family of GTPases also has well-documented functions on vascular smooth muscle contraction, proliferation, or actin cytoskeletal organization. Of particular interest is the role of Rho in "calcium sensitization" of smooth muscle (for review see Ref. 18). The explanation of this phenomenon is that RhoA leads to activation of Rho-dependent kinase and subsequent phosphorylation of the regulatory subunit of myosin light chain phosphatase, producing inhibition of phosphatase acitivity. The result is more phosphorylated myosin light chain and therefore more contraction at the same calcium level. Agonists such as angiotensin II (21) and thrombin (17) have been identified as initiating stimuli for Rho-mediated changes. Whereas activation of purinergic receptors lead to number of changes in vascular smooth muscle, they have not previously been connected with the Rho pathway until a study reported here. Another group of large G protein-coupled receptors are now reported to link with Rho. Sauzeau and colleagues (16) have opened a new area of investigation by showing that the purinergic receptors, P2Y₁, P2Y₂, P2Y₄, and P2Y₆ are coupled to Rho and Rho kinase activation in vascular myoctyes in culture and that this leads to both actin cytoskeletal organization and the induction of calcium sensitization.

Because the small GTPases produce cell growth and proliferation in many different preparations, it is not surprising that they are considered candidates for investigation in conditions of vascular smooth muscle proliferation in injury. Two of the studies in this issue link the Rho pathway to restenosis, which can also be related to cellular growth. Muniyappa et al. (11) show that Rho is inhibitory to the pathway by which interleukin-1 (IL-1) induces the expression of inducible nitric oxide synthase (iNOS). This study builds on earlier data relating IL-1 to the induction of iNOS and a role for NO in reducing neointimal proliferation in injury with implications for restenosis. Eto et al. (3) has also investigated the role of Rho in restenosis. In a pig model, Rho kinase activity was reduced by the introduction of an adenoviral vector encoding a dominant negative form of Rho kinase into the vessel wall after balloon injury. The neointimal formation at the site of injury was suppressed compared with animals into which the -galactosidase was introduced as a control, providing further evidence for a role for Rho in cell proliferation in injury.

The final study addresses the involvement of Rho in signals for muscle differentiation (20). RhoA and integrin are related in focal adhesion formation and stress fiber assembly (review Ref. 8). This provides a rationale for investigating the involvement of integrin in RhoA regulation of skeletal -actin promoter activity. Here, Wei et al. (20) expands on their early studies showing that RhoA regulates expression of skeletal -actin by demonstrating that 1-integrin stimulates Rho-dependent activation of the skeletal -actin promoter. In this process they have also implicated the focal adhesion kinase and phosphatidylinositol 3-kinase.

There remain unresolved questions as to the contributions of each low-molecular-weight GTPase protein to different components of myocardial and vascular hypertrophy and hyperplasia, as well as interactions among the pathways. Differences between the effects in in vivo versus in vitro situations and between cells during development versus mature cells need to be clarified. In addition, the role of the guanine nucleotide exchange factors and GTPase-activating proteins in regulating the small G proteins is only beginning to be explored in relation to cardiovascular disease.

	FOOTNOTES

This special topic section is a collection of papers accepted under a special call for manuscripts by the Editor. See Journal web site for information about the next call.

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TOP ARTICLE REFERENCES

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2.   Aoki, H, Izumo S, and Sadoshima J. Angiotensin II activates Rho A in cardiac myocytes: a critical role of Rho A in angiotensin II-induced premyofibril formation. Circ Res 82: 666-676, 1998[Abstract/Free Full Text].

3.   Eto, Y, Shimokawa H, Hiroki J, Orishige K, Kandabashi T, Matsumoto Y, Amano M, Hoshijima M, Kaibuchi K, and Takeshita A. Gene transfer of dominant-negative Rho-kinase suppresses neointimal formation after balloon injury in pigs. Am J Physiol Heart Circ Physiol 278: H1744-H1750, 2000[Abstract/Free Full Text].

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5.   Gibbs, JB, Sigal IS, Poe M, and Scolnick EM. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci USA 81: 5704-5708, 1984[Abstract/Free Full Text].

6.   Hoshijima, M, Sah VP, Wang Y, Chien KR, and Brown JH. The low molecular weight GTPase Rho regulates myofibril formation and organization in neonatal rat ventricular myocytes. J Biol Chem 273: 7725-7730, 1998[Abstract/Free Full Text].

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9.   Morissette, MR, Sah VP, Glembotski CC, and Brown JH. The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element. Am J Physiol Heart Circ Physiol 278: H1769-H1774, 2000[Abstract/Free Full Text].

10.   Ridley, AJ. Rho family proteins and regulation of the actin cytoskeleton. Prog Mol Subcell Biol 22: 1-22, 1999[Medline].

11.   Muniyappa, R, Xu R, Ram JL, and Sowers JR. Inhibition of Rho protein stimulates inducible nitric oxide synthase expression in rat vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 278: H1762-H1768, 2000[Abstract/Free Full Text].

12.   Pracyk, JB, Tanaka K, Hegland DD, Kim Y-S, Sethi R, Rovira II, Blazina DR, Lee L, Bruder JT, Kovesdi I, Goldshmidt-Clermont PJ, Irani K, and Finkel T. A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy. J Clin Invest 102: 929-937, 1998[ISI][Medline].

13.   Ramirez, MT, Sah VP, Zhao XL, Huner JJ, Chen KR, and Brown JH. The MEKK-JNK pathway is stimulated by ₁ adrenergic receptor and Ras activation and is associated with in vitro and in vivo cardiac hypertrophy. J Biol Chem 272: 14057-14061, 1997[Abstract/Free Full Text].

14.   Sah, VP, Hoshijima M, Chien KR, and Brown JH. Rho is required for G_q- and ₁-adrenergic receptor signaling in cardiomyocytes. J Biol Chem 271: 31185-31190, 1996[Abstract/Free Full Text].

15.   Sah, V, Minamisawa S, Tam SP, Wu TH, Dorn GW, II, Ross J, Chien KR, and Brown JH. Cardiac Specific overexpression of RhoA results in sinus and atrioventricular nodal dysfunction and contractile failure. J Clin Invest 103: 1627-1634, 1999[ISI][Medline].

16.   Sauzeau, V, LeJeune H, Cario-Toumaniantz C, Vaillant N, Gadeau A-P, Desgranges C, Scalbert E, Chardin P, Pacaud P, and Loirand G. P2Y ₁, P2Y₂, P2Y₄, P2Y₆ receptor subtypes are coupled to Rho and Rho-kinase activation in vascular myocytes. Am J Physiol Heart Circ Physiol 278: H1751-H1761, 2000[Abstract/Free Full Text].

17.   Seasholtz, TM, Majumdar M, Kaplan DD, and Brown JH. Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res 84: 1186-1193, 1999[Abstract/Free Full Text].

18.   Somlyo, AP, and Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol (Lond) 522: 177-185, 2000[Abstract/Free Full Text].

19.   Thorburn, J, Xu S, and Thorburn A. MAP kinase- and Rho-dependent signals interact to regulate gene expression but not actin morphology in cardiac muscle cells. EMBO J 16: 1888-1900, 1997[ISI][Medline].

20.   Wei, L, Zhou W, Wang L, and Schwartz RJ. 1 integrin and PI 3 kinase regulate RhoA-dependent activation of the skeletal -actin promoter in myoblasts. Am J Physiol Heart Circ Physiol 278: H1736-H1743, 2000[Abstract/Free Full Text].

21.   Yamakawa, T, Tanaka S, Numaguchi K, Yamakawa Y, Motley ED, Ichihara S, and Inagami T. Involvement of Rho-kinase in angiotensin II-induced hypertrophy of rat vascular smooth muscle cells. Hypertension 35: 313-318, 2000[Abstract/Free Full Text].