doi: 10.1161/01.RES.0000216039.75913.9e
© 2006 American Heart Association, Inc.
Reviews |
The Rac and Rho Hall of Fame
A Decade of Hypertrophic Signaling Hits
From the Department of Pharmacology (J.H.B., D.P.D.R.) and Biomedical Sciences Graduate Program (D.P.D.R.), University of California, San Diego; and San Diego State University Heart Institute and Department of Biology (M.A.S.), San Diego State University.
Correspondence to Mark A. Sussman, San Diego State University, SDSU Heart Institute and Department of Biology, Rm 426, 5500 Campanile Dr, San Diego, CA 92182. E-mail sussman{at}heart.sdsu.edu
This Review is part of a thematic series on The Role of Small GTPases in Cardiovascular Biology, which includes the following articles:
Rho GTPases, Statins, and NO
The Role of Small GTPases in Endothelial Cytoskeletal Dynamics and the Sheer Stress Response
Rho Kinases in Cardiovascular Physiology and Pathophysiology
Regulation of NADPH Oxidases: The Role of Rac Proteins
The Rac and Rho Hall of Fame: A Decade of Hypertrophic Signaling Hits
Rho GTPases and Leukocyte Adhesion Receptor Expression and Function in Endothelial Cells
Anne Ridley Guest Editor
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Abstract |
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 Top Abstract IntroductionRho Family Activation,...RhoARac1Statins and RhoGTPasesConclusionsReferences |
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Over the last decade, the Rho family GTPases have gained considerable recognition as powerful regulators of actin cytoskeletal organization. As with many high profile signal transducers, these molecules soon attracted the attention of the cardiovascular research community. Shortly thereafter, two prominent members known as RhoA and Rac1 were linked to agonist-induced gene expression and myofilament organization using the isolated cardiomyocyte cell model. Subsequent creation of transgenic mouse lines provided evidence for more complex roles of RhoA and Rac1 signaling. Clues from in vitro and in vivo studies suggest the involvement of numerous downstream targets of RhoA and Rac1 signaling including serum response factor, NF-
B, and other transcription factors, myofilament proteins, ion channels, and reactive oxygen species generation. Which of these contribute to the observed phenotypic effects of enhanced RhoA and Rac activation in vivo remain to be determined. Current research efforts with a more translational focus have used statins or Rho kinase blockers to assess RhoA and Rac1 as targets for interventional approaches to blunt hypertrophy or heart failure. Generally, salutary effects on remodeling and ischemic damage are observed, but the broad specificity and multiple cellular targets for these drugs within the myocardium demands caution in interpretation. In this review, we assess the evolution of knowledge related to Rac1 and RhoA in the context of hypertrophy and heart failure and highlight the direction that future exploration will lead.
Key Words: hypertrophy • molecular biology • myocardium • small GTPases • statins
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Introduction |
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TopAbstractIntroductionRho Family Activation,...RhoARac1Statins and RhoGTPasesConclusionsReferences |
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Myocardial signal transduction grows increasingly elaborate and intertwined with each passing year as novel relationships are uncovered. Yet, almost paradoxically, out of the complexity, a unifying theme emerges: the need to regulate cardiac remodeling. Structure and function of the heart are inextricably linked in myocardial biology, and, more often than not, every signaling pathway in the heart emanates from or will eventually cascade into changing cellular and organ morphology. Diverse origins of cardiac remodeling are rooted in altered signaling for survival, metabolism, calcium-handling, proliferation, adhesion, contractile protein modification, vesicle transport, and cytoskeletal structure, to name but a few examples. Because the heart is purposefully dedicated to pumping blood, it comes as no surprise that myocardial signal transduction has pervasive influence over shaping the heart. Moreover, this singular purpose in myocardial design poses interesting challenges when extrapolating the function of signaling molecules previously identified in other cell or tissue types in a completely different context. Such was the case for the small GTPases and the avalanche of basic cell biology that accompanied their discovery in the early 1990s, followed later that decade by investigation of their effects in the myocardial milieu.
The low-molecular-weight or small GTPase (Smg) superfamily consists of more than 100 members which are broadly grouped based on structural similarities into 5 subfamilies: (1) Ras, (2) Rho/Rac/cdc42, (3) Arf/Sar1, (4) Rab, and (5) Ran. All constituents are 20- to 30-kDa monomeric G proteins possessing exquisite specificity for regulation of cell structure, as elegantly demonstrated by exchange of pinpointed residues between Ras superfamily members that correlate with "switch-of-function" mutants exhibiting distinct effects on cytoskeletal remodeling.1 In the context of cardiovascular literature, the Arf/Sar1, Rab, and Ran subfamilies have received a smattering of attention2–4 but, overall, remain relatively understudied and will not be discussed in this review. In contrast, Ras, the first small GTPase linked to cardiac remodeling, is the most extensively characterized of the family in the myocardial setting. Ras-mediated hypertrophic signaling has been recently examined in review, and readers are encouraged to seek out these excellent summaries for more details.5,6 Second to Ras, the Rho/Rac/cdc42 family has received greatest attention for their actions in myocardial cells and, of the 20 known Rho family gene products, the most extensively characterized members in the context of myocardial signaling remain Rac1 and RhoA.
The seminal articles of Ridley and Hall, published in Cell less than 15 years ago,7,8 catalyzed the logarithmic growth of research into the role of RhoA and Rac1 on cytoskeletal organization. In fibroblasts and other non-cardiac cells, these responses include formation of focal adhesions, actin stress fibers, lamellipodia, and membrane ruffles, which translate into a diverse set of cellular events including leukocyte migration, smooth muscle contraction, neurite retraction, and cytokinesis.9–11 The repertoire of responses elicited through these Rho family proteins appeared, at first blush, to be distinct from those elicited by their cousin, the Ras GTPases. At the time, intense interest was centered on the role of Ras as an oncogene, its activation through tyrosine kinase growth factor (epidermal growth factor [EGF], platelet-derived growth factor [PDGF]) receptors, and its coupling to the downstream mitogen-activated protein kinase (MAPK) pathways that regulate cell proliferation and transformation.12 This work provided a framework within which to consider Rho family proteins as signal transducers and to explore how they were activated and what downstream effectors they stimulated. As it turned out, the signaling pathways affected through Rho GTPase activation were not limited to those originally described with respect to the actin cytoskeleton but also included regulation of cell proliferation, gene expression, smooth muscle contraction, ion channel activity, endothelial permeability, reactive oxygen species (ROS) production, phospholipid metabolism, and more.9,13
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Rho Family Activation, Regulation, and Effectors |
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TopAbstractIntroductionRho Family Activation,...RhoARac1Statins and RhoGTPasesConclusionsReferences |
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The articles by Ridley and Hall7,8 were also notable in that 2 of the agonists shown to work through activation of RhoA and Rac1, lysophosphatidic acid (LPA) and bombesin, are ligands for G protein–coupled receptors (GPCRs). Around the same time, LPA effects on phosphatidylinositol (PI) 3-kinase and tyrosine phosphorylation were demonstrated by Narumiya and colleagues14 to be sensitive to the C3 exoenzyme from botulinus toxin, which ribosylates and inactivates Rho.15 Bombesin was shown in the Ridley and Hall studies to act through Rac1 as well as RhoA. In addition chemoattractants such as N-formyl-methionine-leucine-phenylalanine (fMLP), a GPCR agonist with well-characterized effects on neutrophils, elicits Rac1 activation.16 Thus although integrins, adhesion molecules, cytokines, and receptor tyrosine kinases are all known to activate RhoA and Rac1, the effects of GPCR signaling are most remarkable (Figure 1).
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The small GTPases differ from heterotrimeric G proteins (also GTPases) in that they consist of only a single subunit, homologous to but about half the size of the heterotrimeric G-protein 
subunit. The small GTPases do not interact with GPCRs or with ß/
subunits. They become active when GTP is exchanged for GDP; however, the release of GDP is catalyzed not by agonist-bound GPCRs but rather through the actions of guanine nucleotide exchange factors (GEFs). Most of the GEFs that act on RhoA and Rac1 are in the 60-member Dbl family, which is characterized by the proteins containing both Dbl homology (DH) and plexstrin homology (PH) domains. There is a vast and growing literature describing the characteristics of and pathways by which these GEFs are activated.9,17,18 For the purposes of this review, several of the more widely accepted molecular mechanisms of GEF activation will be mentioned. One is the fundamental paradigm demonstrated for activation of Rho GEFs in response to GPCR stimulation, as first described for the p115 Rho GEF19,20 and shown in Figure 1. In seminal articles on p115Rho GEF activation, the heterotrimeric G-protein subunits of the G12/13 family (G12,G13) were shown to bind to and (for G13) stimulate the GTP exchange activity of the p115 RhoGEF. The role of RhoGEFs as effectors for the G12 and G13 proteins, and the mechanism by which GPCRs that efficiently couple to G12/13 (LPA, sphingosine 1-phosphate [S1P], thrombin, thromboxane A2 [TXA2], etc) activate Rho GEFs and subsequently RhoA, is now well accepted. There is also recent evidence that in some systems, Rho GEFs can be activated by the subunits of Gq (Gq), which normally couples to phospholipase C.21–24 For Rac1, prominent mechanisms of GEF activation are elicited through activation of GPCRs that couple to Gi (fMLP, bombesin, endothelin-1 (ET-1), LPA, etc) or through receptor tyrosine kinases. These receptors efficiently stimulate PI3-kinase activity, increasing formation of PIP3, which can activate Rac GEFs such as Tiam 1, P-Rex 1, and others9,25 (Figure 1). Thus, both RhoA and Rac1 can be activated by signaling mechanisms already known to play prominent roles in cardiac remodeling. Two additional types of molecules regulating Rho GTPase activity bear mention. These are the GTPase activating proteins (GAPs), which stimulate GTP hydrolysis and thereby inactivate RhoA or Rac1 and the GDP dissociation inhibitors (GDIs), which prevent GDP from dissociating from the small GTPases and thus inhibit their activation by GEFs.26
The effects of RhoA and Rac1 on the actin cytoskeleton and cell morphology are mediated through stimulation of downstream effector kinases by the activated (GTP-liganded) Rho protein (Figure 2). For RhoA, the best known effectors are Rho kinase (ROCK) and mammalian diaphanous (mDia). The regulation and function of the Rho kinases has been recently reviewed.27 ROCK phosphorylates the myosin binding subunit of myosin light chain (MLC) phosphatase,28,29 resulting in increased myosin phosphorylation and contraction.28 The discovery of a role for RhoA- and ROCK-mediated myosin phosphorylation in vascular smooth muscle contraction,28–30 the finding that RhoA activity is upregulated in hypertension31–33 and the demonstrated efficacy of the ROCK inhibitor Y-27632 in lowering blood pressure34 have convincingly demonstrated involvement of RhoA/ROCK signaling in hypertension (see Lee et al35 for review). RhoA and ROCK signaling pathways have also been implicated in cerebrovascular stroke36 and in vascular endothelial permeability (to be addressed in another section of this thematic series). In contrast to the clear role of RhoA/ROCK pathways in vascular control, the involvement of RhoA/ROCK pathways in cardiac hypertrophy and remodeling is relatively unclear, as discussed later.
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Two predominant effector response pathways lie downstream of Rac1 activation in cellular signaling: induction of cytoskeletal remodeling and formation of ROS (Figure 2). A diverse team of cytoskeletal remodeling proteins are influenced by Rac1 and referred to by acronyms or unintuitive abbreviations including WASP (Wiskott–Aldrich syndrome protein), WAVE (WASP with a V-domain), IQGAP (calmodulin-binding GTPase activating proteins), and PAK (p21-activated kinase).37 PAK shows homology to another kinase that binds to Rac1 named mixed lineage kinase 3 (MLK3) that, on activation, promotes the MEKK-SEK-JNK signaling cascade.38 Our understanding of how cytoskeletal dynamics in the myocardium is regulated by this alphabet soup of factors is very rudimentary, but at least PAK has achieved visibility in regulation of contractility.39,40 The cardiovascular literature is, in contrast, replete with examinations of ROS-mediated effects that can be triggered, at least in part, by Rac1 binding to p67 PHOX, leading to activation of the NADPH oxidase system. The subsequent generation of ROS, which cascades into multiple cardiovascular effects including hypertrophy, hypertension, atherosclerosis, chemotaxis, and platelet aggregation, has been recently summarized41 and is considered elsewhere in this thematic series. The emphasis in this review is placed primarily on Rac-mediated myocardial hypertrophic effects.
Studies using activated or dominant interfering mutants of RhoA or Rac1 have implicated these proteins in numerous pathways regulating gene expression (Figure 2). Among the earliest described effects of RhoA was its ability to elicit serum response element (SRE)-mediated transcriptional activation through a TCF-independent, SRF-mediated pathway.42 In addition, RhoA has been implicated in transcriptional activation of AP-1,43,44 GATA-4,45 nuclear factor (NF)-B,46,47 and MEF2 pathways.48,49 For Rac1, activation of MAPK kinase cascades upstream of c-Jun N-terminal kinase (JNK), and p38 elicits transcriptional changes.50,51 For example, regulation of target genes such as brain natriuretic peptide52 or atrial natriuretic factor (ANF)53 are influenced by Rac1 activity. As discussed below, Rac1-mediated activation of the apoptosis signal-regulating kinase 1 (ASK1) also leads to regulation of NF-B activity. In evaluating the downstream consequences of Rac1 activation, substantial differences in gene profiling were found in comparing Rac1 activity versus other small GTP-binding proteins. The transcriptional profile for Rac1 was most closely mimicked by Cdc42, then RhoA, and shared the least in common with Ras. Genes upregulated by Rac1 in NIH 3T3 cells included participants in cell adhesion and extracellular matrix, whereas some cell cycle genes were downregulated by Rac1 and Cdc42 (and increased by RhoA).54 Thus, there are numerous potential pathways by which RhoA or Rac1 activation and signaling could contribute to the well-described alterations in gene expression associated with cardiomyocyte hypertrophy and remodeling.
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RhoA |
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TopAbstractIntroductionRho Family Activation,...RhoARac1Statins and RhoGTPasesConclusionsReferences |
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In Vitro
There are compelling data indicating that RhoA is required for cell transformation and proliferation in a variety of cell types. Mechanisms by which RhoA synergizes with Ras to allow cell cycle reentry, including downregulation of cyclin-dependent kinases, have been identified in numerous cell lines including fibroblasts and vascular smooth muscle.31,55–57 Because cardiomyocytes are terminally differentiated, growth signals do not result in proliferation but rather in cellular hypertrophy. It is perhaps not surprising then that RhoA has been found to mediate hypertrophic growth of neonatal rat cardiomyocytes, often in synergy with Ras/MAPK activation.
The effects of RhoA on hypertrophy were first investigated in neonatal rat ventricular cardiomyocytes, a tractable system for evaluating changes in cardiac gene expression, protein synthesis, and cell morphology in response to extracellular ligands or transfected gene products. It is well accepted that agonists for receptors that couple primarily to the heterotrimeric G protein Gq, including phenylephrine (PE), ET-1, and prostaglandin F2 (PGF2), elicit robust hypertrophic responses in this model system. Inhibition of Gq signaling blocks, whereas expression of Gq mimics, these hypertrophic effects of agonists.58–60 Responses to PE have also been shown to be mediated through Ras58 and subsequent activation of extracellular signal-regulated kinase (ERK) and MAPK, which have been extensively linked to induction of hypertrophy.61,62 Data implicating RhoA in Gq-mediated hypertrophic responses have come primarily from the use of inhibitors of RhoA signaling. Thus, PE-induced increases in myocyte size and increased protein production appear to be RhoA/ROCK dependent, as they are blocked by RhoA ribosylation with C3 toxin and by Rho kinase inhibition with Y-27632.63,64 PE-induced transcriptional activation of ANF, myosin light chain 2V (MLC-2), and -myosin heavy chain (-MHC) can also be inhibited by treatment with C3 or Y-27632 or by overexpression of dominant-negative mutant (N19) of RhoA.64–66 Finally, the characteristic cytoskeletal response evidenced by myofilament organization is prevented by C3.63 Similar conclusions have been drawn from studies using ET-1 or angiotensin (Ang) II, both agonists for GPCRs that couple robustly to Gq. Changes in cell size, protein production, gene transcription, and myofibril organization elicited by ET-1 are blocked by a variety of RhoA/ROCK inhibitors including C3 toxin, Y-27632, and dnRhoA constructs.45,67 Blocking RhoA signaling with C3 is also able to prevent Ang II–induced myofilament organization and ANF expression.68
Additional studies using expression of activated Gq to induce ANF, AP-1, and MLC-2 promoter-luciferase activity demonstrate that these responses to Gq and PE are inhibited by coexpression of a dominant-negative RhoA construct or by treatment with C3 toxin.69 These data further support a role of RhoA in Gq-induced hypertrophy. Inhibition of Ras was also shown to inhibit Gq- and PE-induced hypertrophy in this system. Subsequent studies from our laboratory demonstrated that adenoviral expression of a constitutively activated mutant (L63A) of RhoA was able to upregulate hypertrophic gene expression and induce myofilament organization through a Rho kinase–dependent pathway.66,70 Our data also suggested that RhoA and the small G protein Ras worked in concert to transactivate genes associated with hypertrophy. Thus expression of activated forms of Ras and RhoA increased ANF reporter gene activity synergistically,70 and dominant-negative RhoA did not block the hypertrophic effects of activated Ras.66 Thorburn’s laboratory also showed that dnRas and dnRhoA constructs reduced Gq-induced hypertrophic gene expression in an additive fashion.69 These data suggest that the Ras/MAPK and RhoA signaling pathways contribute in parallel to hypertrophic responses in neonatal rat cardiomyocytes.
An important recent development that enabled the study of RhoA signaling was the design and dissemination of an assay for directly quantifying RhoA activation.71 This Rho pull-down assay is based on the selective ability of GTP-liganded Rho to tightly associate with the Rho binding domain (RBD) of its effector, rhotekin. Applying this assay to studies of RhoA activation in neonatal rat cardiomyocytes, it is apparent that the agonists that are most efficacious for inducing hypertrophy are not those that are best at activating RhoA. Phenylephrine effects on RhoA activation have been assessed using the RhoA pull-down assay71 and indicate 2- to 3-fold increases in response to this agonist.72,73 ET-1 is, by comparison, somewhat more effective,73 whereas Ang II, which some contend to be without direct hypertrophic effects on cardiomyocytes, increases RhoA GTP binding to a modest extent.74 Both of these ligands activate GPCRs that regulate Gq but also appear able to activate G12/13.75 Most remarkable are the effects of LPA76 and S1P (C.K. Means and J.H.B., unpublished data, 2004), which elicit 6- to 10-fold increases in the amount of activated RhoA in cardiomyocytes. The ability of LPA, thrombin, and S1P to robustly activate RhoA is consistent with the literature indicating that the receptors for these ligands efficiently and often selectively couple to G12/13.75
Work from our laboratory has shown that stimulation of neonatal myocytes with LPA induces hypertrophic gene expression, cytoskeletal rearrangement, and ERK activation73; however, this response is not as robust as that elicited by the more commonly studied Gq coupled receptor agonists. Indeed LPA activates Gq pathways relatively poorly, inducing only modest increase in phosphoinositide hydrolysis.73 The model suggested for LPA-induced hypertrophy is one in which there is again parallel activation of Ras (in this case, mediated through Gi signaling) and RhoA (through G12/13). There is also evidence that PE can activate RhoA-dependent protein synthesis and cytoskeletal changes in cardiomyocytes through G12/13.63 Finally, expression of activated G13 in cardiomyoctyes has been shown to induce ANF promoter activity and increase myocyte size in a RhoA-dependent manner.76 On the other hand, PE and ET-1 use Gq or Gi signaling pathways in adult cardiomyocytes but, in contrast to thrombin, do not signal through G12/13 to activate ERK.77
Stretch has been shown to activate fetal gene expression, increase protein synthesis, induce myofilament organization, and activate MAPK signaling pathways, indicative of myocyte hypertrophy.78–80 Like GPCR stimulation, stretch can activate RhoA and Rac1.80,81 Our laboratory used this model of stretch-induced cardiomyocyte hypertrophy to demonstrate that RhoA and Rac1 activation in response to stretch appears localized to, and dependent on, caveolae. One role for RhoA activation, as suggested by this work, is through its effects on the actin cytoskeleton. RhoA-mediated actin cytoskeletal rearrangement appears to be necessary for the translocation of activated ERK to the nucleus,81 providing a mechanism for synergy between the Ras/ERK and RhoA pathways regulating gene transcription and inducing hypertrophy.
Specific mechanisms by which RhoA contributes to cardiomyocyte hypertrophic gene expression are suggested by the plethora of transcriptional mediators downstream of RhoA. For example, RhoA can regulate muscle specific gene expression through activation of serum response factor (SRF). Numerous reports from the laboratories of Treisman, Schwartz, and others document the involvement of Rho family proteins in SRF-dependent gene transcription in a variety of cell types.42,82–85 Both wild-type and activated forms of RhoA can regulate SRF activity by increasing SRF-dependent promoter expression in smooth muscle cells and myoblasts. Expression of dnRhoA, as well as C3 and Y-27632 treatment, have been shown to decrease basal SRF driven promoter activity, suggesting that RhoA may regulate SRF basally.84–86 Inhibition of Rho kinase also inhibited SRF–DNA binding and decreased nuclear content of SRF in smooth muscle cells.85 Results from our laboratory demonstrate that an activated mutant of protein kinase N (PKN), a known RhoA effector, can induce ANF expression and suggest that it may also be involved as an upstream modulator of SRF in RhoA-mediated hypertrophic gene expression.87 In light of the requirement for SRF in induction of ANF and other cardiac hypertrophic genes,88 it is likely that SRF is an important target of RhoA signaling in the cardiac context.
Other transcriptional targets of importance to cardiomyocyte hypertrophy are potential RhoA targets. Transcriptional activation by GATA-4, a known regulator of hypertrophic gene expression in cardiomyocytes, is potentiated by coexpression of activated RhoA. The mechanistic basis for this is not clear, although it appears that the effect of RhoA relies on p38 MAPK-dependent phosphorylation of GATA-4.45 Rho kinase activation could also be involved, as a recent report demonstrated that expression of dnROCK prevents GATA-4 responsive promoter-driven luciferase expression in cardiomyocytes.64 Treatment with Y-27632 was also found to decrease PE induced increases in GATA-4 DNA binding, further implicating RhoA/ROCK signaling in the regulation of GATA-4.64 In a recent report from the laboratory of Olson, several components of the Rho family signaling pathway were identified in a screen for HDAC5 regulation.49 Candidates include RhoA, RhoC, and 2 Rho-specific nucleotide exchange factors, suggesting possible involvement of RhoA activation and signaling in the regulation of HDAC function and thereby MEF2 transcriptional activity. Previous work in myoblasts also implicated RhoA in MEF2 mediated gene expression.48 There are also data, albeit not in cardiomyocytes, demonstrating that RhoA is involved in regulating c-Jun and c-Fos expression and can influence AP-1 mediated gene expression.43,44,86,89 Finally, and importantly, RhoA signaling pathways have been implicated in the activation of NF-B.46,47 Although this has not yet been demonstrated to occur in cardiomyocytes, classic hypertrophic agonists such as PE, Ang II, and ET-1,90 as well as tumor necrosis factor (TNF)-,91 have been shown to activate NF-B in cardiomyocytes, and a potential role for NF-B as a therapeutic target in hypertrophy has been considered.92
In Vivo
Several transgenic and knockout mouse models have been examined to delineate the role of RhoA signaling pathways in vivo. In work from our laboratory, Sah et al93 generated transgenic mice in which constitutively activated (L63A) RhoA or wild-type RhoA were expressed in a cardiac specific manner through the -MHC promoter. Most founders and progeny of the activated RhoA lines did not survive to adulthood. Mice expressing wild-type RhoA also showed increased mortality, which was dependent on gene dose and modestly rescued by backcrossing into a C57 (versus Black Swiss) line to give 50% survival at approximately 3 months. Surprisingly, these mice did not develop an obvious hypertrophic response. Although both ANF and -MHC expression were increased, there was no significant increase in ventricular weight to tibial length, nor was there an increase in cardiomyocyte size. On the other hand, evidence of heart failure was seen in RhoA transgenics, with severe edema, ventricular chamber dilation, increased cardiac fibrosis, atrial enlargement, and decreased fractional shortening. It is not clear what underlies the failure of in vivo RhoA expression to mimic the in vitro hypertrophic phenotype, but effects on postnatal development, compensatory responses, or the more prolonged duration of RhoA expression are possibilities. It is also notable that a recent study using animals in which the RhoA effector, ROCK1, was partially deleted (ROCK1 haploinsufficient mice) did not show decreased hypertrophy but rather decreased fibrosis.94 Applying these data to the observations made with the cardiac RhoA transgenic mouse suggests the possibility that RhoA in cardiomyocytes could act through a ROCK-dependent paracrine pathway to alter cardiac fibroblast proliferation. Another striking observation made in the RhoA transgenic mice was that many developed atrial fibrillation and atrioventricular (AV) block, as well as severe bradycardia, with heart rates nearly half those of nontransgenic littermate controls.93 Effects on the conduction system could reflect effects of RhoA in modulating the activity of cardiac ion channels.95,96
A different transgenic line expressing RhoGDI behind the -MHC promoter was generated in the Schwartz laboratory. The expected effect of enhanced RhoGDI expression would be the sequestration of inactive Rho proteins in the cytosol, and indeed these transgenic mice exhibited decreases in the amount of RhoA, Rac1, and Cdc42 localized in the membrane fraction. Notably, however, expression of these proteins was also markedly upregulated, with total and cytosolic protein levels increased roughly 10-fold. This is likely a compensatory feedback mechanism resulting from the Rho GTPases being locked in the cytoplasm and rendered nonactivatable. Whether the observed phenotype derives from the decreases in Rho proteins at the membrane where they are normally activated, or from the greatly enhanced amounts of RhoA or Rac1 in the cytoplasm, where some could be constitutively activated, is unclear. Progeny of the higher copy number lines showed defects in cardiac development associated with reduced myocyte proliferation and embryonic lethality.97 Lines with lower copy number used in subsequent studies98 showed atrial enlargement and mild ventricular hypertrophy by 4 months. The predominant phenotype of the RhoGDI mice resembled that of the RhoA transgenic, ie, a progressive AV conduction defect and a decrease in heart rate. The authors suggested that the concomitant down regulation of the gap junction protein connexin 40 could explain their observations.96 The AV and sinoatrial (SA) nodal defects seen in the RhoA transgenic and in the RhoGDI transgenic mice could also result from effects of RhoA or Rac1 on ion channels in cardiac SA nodal and conduction tissue.
A more fortuitous set of findings emerged from the characterization of mice with germline deletion of a GDP dissociation stimulator (Smg GDS) that functions like a GEF.99 Lack of GEF function would be predicted to limit the ability for either RhoA or Rac1 activation and signaling. Postnatal survival of the homozygous knockout mice was severely impaired, and neonatal death was associated with myocardial changes including thinning of the atrial, ventricular, and septal walls. Marked increases in apoptosis were observed by TUNEL staining of embryonic hearts from GDS–/– versus GDS–/+ or wild-type mice. No changes in cardiomyocyte proliferation, gene expression, or differentiation were evident. The authors note that this Smg GDS also regulates Ki Ras; thus, loss of Ras activation could contribute to decreased cell survival. More intriguing is the implication from these data that loss of RhoA signaling leads to apoptosis. There is evidence that Rho proteins play a protective role in cell survival in other cell types, ie, RhoA inactivation enhances apoptosis in baby hamster kidney cells100 and Rac1 affects apoptosis in BaF3 cells.101 There are in fact several protective pathways that could be activated by RhoA expression. Although not examined in cardiomyocytes, these include RhoA-mediated regulation of PI3- and PI4,5-kinase, Akt, PTEN, and NF-B.14,46,47,102–105
RhoA/ROCK expression and activity are increased in a variety of disease models, including hypertension, myocardial infarction, and pressure overload.106–109 Whether this response is ultimately beneficial or detrimental to cardiac performance and survival remains to be further clarified. Several recent studies have looked, albeit indirectly, at the role of RhoA signaling in the response to ischemia/reperfusion or infarction. In these studies, the Rho kinase inhibitors, Y-27632 and fasudil, were shown to decrease myocardial fibrosis in response to myocardial infarction and in a chronic hypertension rat model of congestive heart failure.106,108 Long-term inhibition of Rho kinase with fasudil treatment, beginning 1 day after coronary artery ligation and continuing for the duration of the 4-week experiment, prevented increases in myocardial fibrosis in mice.108 In a rat model of congestive heart failure, treatment with Y-27632 for 7 weeks blocked the increased myocardial fibrosis and development of failure; increases in RhoA and Rho kinase expression were also prevented.106 Inhibition of ROCK with Y-27632 was also shown to reduce infarct size and apoptosis resulting from 30 minutes ischemia followed by 150 minutes (rat) or 24 hour (mouse) of reperfusion.110,111 Interestingly, in the mouse ischemia/reperfusion model, inhibition of Rho kinase prevented the ischemia/reperfusion-induced increase in proinflammatory cytokines seen with 24-hour reperfusion, suggesting a possible role for RhoA/ROCK signaling in inflammation in vivo.111
The implication of these findings is that activation of RhoA/ROCK signaling pathways in the intact heart is deleterious. What is unclear from these studies, however, is whether the observed salutary effect of blocking RhoA/Rho kinase signaling reflects changes in the cardiomyocyte or is a consequence of more directly inhibiting fibroblast proliferation/migration or of attenuating local inflammatory responses. There is evidence that Rho can be activated in cardiac fibroblasts and affect their proliferation.112 Indeed the study cited above using ROCK 1 haploinsufficient (–/+) mice supports the notion that a critical function for RhoA/ROCK1 could reside in the fibroblast where it mediates perivascular fibrosis induced by TAC, myocardial infarction, or Ang II. Determining whether acute activation of RhoA and ROCK signaling pathways within the myocyte serve protective or deleterious functions is critical to understanding the utility, timing, and targets of inhibition of RhoA signaling.
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Rac1 |
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TopAbstractIntroductionRho Family Activation,...RhoARac1Statins and RhoGTPasesConclusionsReferences |
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In Vitro
As with RhoA, virtually all studies related to Rac1-mediated effects in myocardial cells have relied on neonatal rat ventricular cardiomyocytes. The prevailing cell biology literature had established that Rac family members stimulate formation of lamellipodia and membrane ruffles in cultured fibroblasts.7–11 However, these observations do not readily extrapolate to the architecturally complex cytoskeletal organization of primary cardiomyocyte cultures. Researchers followed the precedent of Ras-mediated hypertrophy, reasoning that other small GTPase family members would be influenced by hypertrophic stimuli and exert influence on hypertrophic remodeling. This postulate was borne out in studies using mutant Rac1 expressed either as constitutively active (V12) or dominant-negative (N17) forms that, when introduced into neonatal cardiomyocyte cultures, produced morphological and phenotypic effects consistent with a role for Rac1 regulation of hypertrophy. Adenoviral-mediated overexpression of V12Rac1 prompted hypertrophic remodeling indistinguishable from that induced by traditional ligand stimulation, whereas the inhibition of Rac1 signaling with the N17Rac1 construct efficiently blocked effects of hypertrophic agonist treatment.113
As an officially anointed member of the molecular signaling intermediates that regulate hypertrophic remodeling of cardiomyocytes, subsequent studies established the context for Rac1-mediated hypertrophic effects and its relationship to MAPK cascades. Typical hypertrophic agonists such as PE and ET-1, were shown to activate Rac1, and the ability of these agonists to induce ERK activity was inhibited by N17Rac1.72 Coupled with the observation that V12Rac1 promotes ERK activation, Clerk et al72 concluded that Rac1 connected hypertrophic stimuli to the MAPK cascade through modulation of ERK and possibly JNK, but not p38. In another study, V12Rac1 increased p38 MAPK activation and stretch-induced hypertrophy led to p38 MAPK activation along with concomitant increases in protein synthesis that are inhibited by N17Rac1.114 Caveolar localization is critical for Rac1 activation in the mechanical stretch model,81 supported by additional studies using Ang II stimulation, which linked trafficking with coincident activation of Rac1 and AT1 receptors into caveolae/lipid rafts.115 Rac1 has also been linked to activation of JNK in cultured cardiac myocytes116 or fibroblasts,51,111,117 with the provocative stimuli in these studies originating from reoxygenation or Ang II treatment, respectively.
Treatments including mechanical stretch, reoxygenation damage, ischemia/reperfusion injury, and Ang II treatment all promote formation of ROS. In light of considerable evidence linking Rac1 activation to ROS generation, this created an area of signaling notoriety for Rac1. Connections among ROS, hypertrophy, and heart failure118,119 and the potential for salutary effects resulting from blocked ROS production120,121 propelled Rac1 into scrutiny. For example, neonatal rat ventricular myocytes were shown to be protected from ROS generation resulting from reoxygenation damage by overexpression of N17Rac1.121 Hypertrophic stimulation by mechanical stretch induces intracellular ROS generation that can also be inhibited by N17Rac1114 and Ang II-induced ROS production associated with Rac1 activation that is inhibited by N17Rac1.53,116 Recent studies suggest Ang II–induced ROS production is dependent on Rac1 activation that cascades into JNK and p38, but not ERK, signaling.74 Relationships between Rac1 and oxidant stress in cardiomyocytes await further clarification, but tantalizing clues from other cell types could help point the way: in hepatocytes, the prosurvival Akt signaling pathway suppresses Rac1-regulated ROS production122; whereas, in endothelial cells, Rac1 regulates the production of the potent antioxidant manganese superoxide dismutase (MnSOD).123
Statins block the isoprenylation and thus the membrane targeting and functional activation of Rho family members. Inhibition of Rac1 and RhoA by statins reduces myocardial expression of hypertrophic markers such as atrial natriuretic factor and myosin light chain 2V in response to Ang II exposure.124 In the case of Ang II-stimulated cardiomyocytes, simvastatin inhibited hypertrophy was linked to decreased Rac1 activity and ROS generation.53 ß-adrenergic stimulation of cultured adult cardiomyocytes with norepinephrine increased Rac1 activation concomitant with apoptotic cell death and this was inhibited by overexpression of N17Rac1 or by treatment with cerivastatin.125 Based on these observations, speculation about the potential of statins for treatment of cardiac hypertrophy and failure in the clinical setting was pursued by further studies using in vivo models (discussed below).
The Rac1/ROS connection to hypertrophy is also manifest in the guise of ASK1 and NF-B. Deconstructing the hypertrophic signaling pathway using a "back-to-front" rationale, Higuchi et al posit that hypertrophy requires NF-B activity, that NF-B activation is facilitated by ASK1 activity, and that ASK1 activation is sensitive to ROS production, thereby leading to the speculation that this entire signaling cascade of events lies downstream from Rac1 activity.126 Indeed, adenoviral-mediated overexpression of V12Rac1 enhanced expression of a NF-B reporter construct and induced degradation of IB, an inhibitor of NF-B activity. In this same study, overexpression of degradation-resistant IB inhibited V12Rac1-driven hypertrophic responses such as increased protein synthesis, atrial natriuretic peptide production, and enhanced sarcomere organization. Moving further upstream in the chain of events, V12Rac1 activates ASK1 and dnASK1 inhibits V12-Rac effects. Conversely, N17Rac1 attenuated cardiac myocyte hypertrophy induced by PE stimulation with attendant blunting of both ASK1 and NF-B activity. And finally, V12Rac1 was unable to initiate the cascade of events leading to hypertrophy when cells were exposed to N-acetyl cysteine, an antioxidant. Thus, the trail can be followed from Rac1 activation through ROS production, subsequent ASK1 and NF-B activation, eventually culminating with myocyte hypertrophy.
Another effector downstream of activated Rac1 is PAK, which has been implicated in alterations of cardiomyocyte contractile mechanics. Activated PAK localizes to sarcomeric Z-disk, interacts with protein phosphatase 2a, and reduces phosphorylation of cardiac TnI and MyBP-C, with these posttranslational modifications influencing contractility by altering calcium sensitivity.40 TnI as well as TnT have been shown to be phosphorylated by ROCK2 and thus presumably through activation of RhoA signaling pathways.127 The phosphorylation of cTnT by ROCK2 results in inhibition of Ca++ activated tension development in skinned fibers. Hypertrophic effects of Rac1 (and RhoA) appear morphologically distinct from those exerted by Cdc42, a closely related Rho family member,128 suggesting specialization of remodeling specification exists between members of the Rho family.
The aforementioned Rac1-mediated effects have little obvious connection to the actin cytoskeleton, where the impact of Rac1-mediated remodeling was first recognized and appreciated. At present, the sole evidence that Rac1 effects on the cytoskeleton are related to hypertrophy is found not in cardiomyocytes, but in microtubules of the vasculature. In vascular smooth muscle cells, Ang II promotes Rac1 activation and this process requires intact microtubule function.129 Indeed Rac1 has been found to bind to tubulin,130 and many studies of Rac1 in noncardiac tissues implicate microtubules as important participants in mediating activation or remodeling.131 As is shown in the next section, there is reason to speculate that microtubules may be involved with Rac1-mediated effects in vivo within the myocardium as well.
In Vivo
Demonstration of Rac1-mediated hypertrophic remodeling in vitro set the stage for researchers to confirm and extend cell culture studies using animal models. Unfortunately, Rac1 cannot be readily studied by conventional gene targeting approaches, because deletion results in embryonic lethality.132 Instead, genetic manipulation of Rac1 to study the consequences of increased myocardial Rac1 activity was accomplished by cardiac-specific transgenesis of V12Rac1.133 Characterization of the Rac1-expressing transgenics revealed an unusual dichotomy: (1) rapid-onset, high-level postnatal expression that led to lethal dilated cardiomyopathy; or (2) slow-onset postnatal expression leading to transient hypertrophy in juvenile mice that resolved with age. The hypertrophic hearts showed no evidence of myofibril disarray and possessed hypercontractile hemodynamic function. Downstream from Rac1 signaling in the myocardium, evidence for activation of PAK and Src was noted in the transgenic hearts. Connections between Rac1 and PAK with respect to myocardial signaling and contractile protein modification were discussed above.39,40 Although the myocardial relationship remains obscure, Rac1 and Src have been linked in a signaling cascade leading to cellular transformation,134 with the biological manifestation of oncogenic signaling in cardiomyocytes typically leading to hypertrophy. The ultimate basis for the observed Rac1-mediated cardiomyopathic phenotypes, like that seen in many cardiac-specific transgenic models based on the -MHC gene promoter, is likely to be a combinatorial effect of postnatal development, transgene expression, and concurrent reactive signaling mechanisms. In a recent follow-up study of the Rac1 transgenic dilated hearts subjected to proteomic analyses, significant increases in both MnSOD and tubulin were identified.135 These are intriguing candidates to be impacted by Rac1 activity because of established precedents linking Rac1 activity to tubulin130,131 and the protective antioxidant effects of MnSOD under conditions of oxidative stress.136,137 MnSOD gene expression and activity are increased in the failing human heart, presumably in response to higher oxidative stress,138 so the elevation of MnSOD in the Rac1 transgenic hearts likely stems from a compensatory response to increased oxidative stress exacerbated by both transgene activity and cardiac failure. ROS levels in Rac1 transgenic hearts have not been determined, so the connections between Rac1, ROS, and hypertrophic remodeling in this experimental paradigm remain to be determined.
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Statins and RhoGTPases |
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TopAbstractIntroductionRho Family Activation,...RhoARac1Statins and RhoGTPasesConclusionsReferences |
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The geranylgeranylation of Rho GTPases (and the farnestylation of Ras) are prevented when isoprenoid synthesis is blocked by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. By inhibiting Ras or Rho isoprenylation, statins can decrease their membrane targeting and activation. Salutary cardiovascular effects of statins that are independent of cholesterol lowering have increasingly been ascribed to alterations in RhoA and Rac1 signaling pathways. Rats subjected to experimentally induced hypertrophy resulting from Ang II treatment or pressure overload and subsequently treated with simvastatin showed inhibition of hypertrophic remodeling. The observed improvement was related to decreased Rac1 activity and lower ROS levels compared with identically challenged control animals.53 These findings are consistent with evidence from failing human heart tissues, which show upregulation of ROS release associated with increased Rac1 activity that can be inhibited by prospective statin treatment.139 Hearts from spontaneously hypertensive rats treated with atorvastatin also showed decreases in cardiac Rac1, concomitant with decreased RhoA activity and downregulation of atrial natriuretic peptide and myosin light chain 2V expression.124 In other studies, simvastatin treatment was shown to prevent pressure overload–induced cardiac hypertrophy, and this was associated with decreases in Ras and ERK activation, as well as with decreased RhoA activation and p27 downregulation.140 Recent studies in a transgenic rabbit model of hypertrophic cardiomyopathy showed prevention of cardiac hypertrophy by atorvastatin, which was associated with reduced levels of membrane Ras and ERK activation without decreases in GTP-bound or total RhoA or Rac1.141 It is therefore clear from these studies that statin treatment can decrease cardiac hypertrophic remodeling, but it is also evident that multiple molecular events may underlie this effect. In addition to possible effects of the statins on processing of Rac1, RhoA, and Ras, the specific cellular site of in vivo statin treatment cannot be ascertained. As the Rho and Ras small G proteins likely function in fibroblast proliferation, neutrophil migration and endothelial cell barrier function, as well as within the cardiomyocyte, there are myriad possible mechanisms by which statins could cause functional improvement. These include alterations in endothelial cell nitric oxide signaling, as fully described in an earlier review in this series.142 The demonstrated involvement of RhoA and Rac1 as signal transducers in cardiomyocytes hypertrophy makes them attractive targets for pharmacological inhibition by statin therapy.143,144 However, as discussed above for studies with Rho kinase inhibitors,106,108,110,111 the development of rational therapeutic strategies will depend on dissecting the roles of these small G proteins in specific cardiac cellular compartments and at various times in the pathophysiological process.
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Conclusions |
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TopAbstractIntroductionRho Family Activation,...RhoARac1Statins and RhoGTPasesConclusionsReferences |
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The last decade has seen considerable progress in our understanding of the relationship of Rac1 and RhoA to cardiomyocyte signaling and hypertrophy. Numerous inciting stimuli, signaling interconnections, downstream targets, effector molecules, and even potential sites for pharmacological intervention have been uncovered. Current studies have focused predominantly on pathophysiological conditions such as inflammatory disease, hypertension, or decompensated failure in which chronic stimulation of Rho or Rac signaling would occur as a result of exposure to activating ligands. In vitro overexpression systems, as well as experimental animal paradigms (transgenics, knockouts), can bypass the subtleties of ligand-activated signaling cascades that often react dynamically and transiently. Thus, the physiological or housekeeping roles of Rac1 and RhoA in maintenance of cardiac homeostasis (eg, the contribution of these GTPases to adaptive physiological hypertrophy induced by exercise) are not yet known. Ongoing actin cytoskeletal remodeling mediated by small GTPases may be an important part of myocardial adaptive responses that remains to be explored. Although there are many gaps in our existing knowledge of these key areas, there are also innovative and provocative data suggesting roles for small GTPases outside the mature myocardium. For example, a study of embryonic stem cells showed that V12Rac1 could either inhibit or promote cardiogenic differentiation depending on the timing of expression relative to cell commitment145 and that N17Rac1 could block cardiogenic differentiation. Thus, there are likely to be important undiscovered relationships between Rac1 and cardiac lineage commitment of stem cells lying in wait. RhoA and ROCK1 have been recently implicated in early cardiac development mediated through the planar polarity pathway and are coexpressed in the outflow myocardium.146 There is also abundant evidence from other systems that RhoA and Rac1 can mutually regulate the activation of the other9,147 and that Rac1-mediated effects can be are antagonized by RhoA,148 but analogous myocardial interactions remain to be explored. Such studies will likely be facilitated through use of conditional/inducible cardiac-specific overexpression or knockout for Rho family members, because use of knockout approaches have provided a wealth of information of the functions of Rho family members in T-cell biology.149 Increasingly sophisticated maneuvers such as these will allow us to look even deeper into the biology of Rho family members and to discover ways to modulate these critical but pleiotropic signal transducers so as to achieve selective therapeutic interventions for cardiac diseases.
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Acknowledgments |
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Support for this article was provided by NIH awards to J.H.B.and M.A.S.
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Footnotes |
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Original received November 28, 2005; resubmission received January 24, 2006; accepted February 3, 2006.
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References |
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