Small Guanine Nucleotide-Binding Proteins and Myocardial Hypertrophy

Ras Subfamily

Several Ras family proteins are readily detected in primary cultures of neonatal rat ventricular myocytes, including HRas, NRas, KRas, and Rap1¹¹ (and unpublished data, A.C. and P.H.S., 1999). Transient transfection with a V12Ras expression vector or microinjection of the protein induces hypertrophic gene expression and increased cell profile.^{12 13 14} Although microinjection of V12Ras protein or adenoviral infection of the V12ras gene may induce myofibrillogenesis,^{12 15} this is not observed in transfection studies.^{12 14} The reasons for this are unclear but may reflect levels of V12Ras expression. Inhibitory N17Ras prevents PE-induced hypertrophic changes.¹² However, its effects are mediated through interaction with Ras.GEFs, which are promiscuous with respect to the activation of small G proteins.¹⁶ Thus, some responses inhibited by N17Ras may be mediated by non-Ras small G proteins.

Rho Subfamily

RhoA and Rac1 are implicated in cardiac myocyte hypertrophy. Transfection or infection of activated RhoA stimulates ANF expression^{15 17 18} and myofibrillogenesis.^{15 19} Dominant inhibitory mutants of RhoA prevent PE-stimulated,^{17 20} G_q-stimulated,²⁰ or Ras-induced²⁰ hypertrophy. Furthermore, C3 exoenzyme, a clostridial toxin that selectively inhibits Rho, inhibits ANF expression induced by hypertrophic stimulation.^{17 18 19 20} Because Rho is implicated in the regulation of the actin-myosin cytoskeleton,⁴ studies have focused on its role in myofibrillogenesis. V14RhoA promotes formation of myofibrillar structures,^{15 19} and in some studies C3 exoenzyme inhibits this process.^{15 19} However, others have failed to detect an effect of C3 exoenzyme on actin myofilaments.¹⁸ These discrepancies may reflect the extent of exposure to the toxin or route of administration. The effects of RhoA on the myofibrillar apparatus may be secondary to modification of the nonmyofibrillar cytoskeleton. Thus, electroporation of C3 exoenzyme into myocytes causes disassembly of focal adhesions and loss of β-actin nonstriated fibrils.²¹

As with V12Ras or V14RhoA, transfection of cardiac myocytes with V12Rac1 increases expression of ANF and BNP.²² Adenoviral infection of myocytes with V12Rac1 also increases ANF expression and promotes morphological changes associated with myocyte hypertrophy, and infection with N17Rac1 inhibits PE-induced hypertrophy.²³ These results implicate both RhoA and Rac1 in cardiac myocyte hypertrophy.

Mechanisms of Activation

The classical Ras isoforms are rapidly activated in myocytes exposed to Ang II, ET-1, and PE.^{11 24} These agonists stimulate protein kinase C (PKC) through G_q/11PCRs, the G_q/11 heterotrimeric G proteins, and phospholipase Cβ.¹ Phorbol esters, which activate PKC directly and induce myocyte hypertrophy,¹ also stimulate Ras.GTP loading.¹¹ Conversely, inhibition of PKC reduces Ras.GTP loading by GPCR agonists.¹¹ These findings suggest that the activation of Ras by Ang II, ET-1, or PE is PKC dependent. However, other PKC-independent signaling molecules (eg, the pertussis toxin-sensitive G_i/o heterotrimeric G proteins) may also be involved.¹¹ It is not clear how GPCR-mediated signaling couples to Ras. As with receptor protein tyrosine kinase stimulation of Ras, adapter proteins (GRB2, Shc) may be involved, which activate the Ras.GEF Sos.²

Activation of Rho subfamily proteins in cardiac myocytes is not well characterized. Ang II induces RhoA translocation to the particulate fraction, consistent with its activation.¹⁹ ET-1 and PE stimulate GTP loading of RhoA and Rac1 (A.C. and P.H.S., unpublished data, 1999). The mechanisms involved are not clear, but in other cells, Rac1 activation is dependent on PI3K,³ which is itself a Ras effector (see below). Additionally, Ras.GEFs are promiscuous and may stimulate GTP loading of several small G proteins.¹⁶

Coupling of Ras to Hypertrophy

Ras binds to and activates several signaling proteins, including c-Raf (MAPK kinase kinase of the ERK cascade), PI3K, and Ral.GDS (GEF for the Ras-like protein Ral) (Figure 2⇑).² The ERK cascade is implicated in hypertrophy, as reviewed recently.¹ Thus, potent hypertrophic stimuli (eg, ET-1, PE, and phorbol esters) activate the ERK cascade, activated components of the cascade stimulate ANF expression and increase myocyte cell profile, and dominant inhibitory components attenuate agonist-stimulated responses. However, not all agonists that activate ERKs induce hypertrophy, and there is little evidence that ERKs promote myofibrillogenesis. Adenoviral infection of signaling molecules has generally been more successful in promoting myofibrillogenesis, but such experiments have not been reported for ERK cascade components. It is probable that additional signaling pathways are necessary for myofibrillogenesis. Studies with V12Ras double mutants that couple selectively to c-Raf, PI3K, or Ral.GDS indicate that c-Raf and Ral.GDS specifically promote hypertrophic gene expression, whereas PI3K has a more global effect.¹⁴ PI3K may act at the level of protein synthesis (reporter genes are transcribed and must be translated for detection), because signaling from PI3K promotes protein synthesis in cardiac myocytes.^{1 25} Indeed, increased protein synthesis is a hallmark of hypertrophy, and PI3K inhibitors suppress protein synthesis in cardiac myocytes.¹

Some researchers propose that Ras-mediated activation of the JNK cascade is more relevant to hypertrophy.²⁴ One line of evidence is that JNK activity is increased, albeit minimally (50%), in hypertrophied hearts of the V12Hras transgenic mouse with no increase in ERK activity. Because these animals were examined at 6 weeks (a considerable period of time subsequent to the initiating hypertrophic signals), JNK activation may reflect the stress of progression from compensated hypertrophy into heart failure. Indeed, there is evidence from patients with heart failure subsequent to ischemic heart disease that this is the case.²⁶ We have previously reviewed the evidence implicating JNKs in hypertrophy,⁶ and our view is that this pathway is more probably associated with apoptosis.

One function of MAPKs is to modulate gene transcription by phosphorylation of transcription factors (eg, Elk1, c-Jun, and ATF2), altering their transactivating activities.¹ Consistent with this, ERKs and JNKs are active in the nuclei of myocytes after stimulation,^{1 11} and c-Jun and ATF2 are both phosphorylated in myocytes subjected to cellular stress or exposed to hypertrophic stimuli.¹

Coupling of RhoA and Rac1 to Hypertrophy

The signaling pathways activated by RhoA to promote hypertrophy are not understood. In other cells, RhoA regulates cell morphology and contractile activity,⁴ and, because myocyte hypertrophy involves changes in cell shape, cytoskeletal modifications are likely to occur. RhoA activates several protein kinases,³ two groups of which have been identified: protein kinase N/PKC-related kinases and Rho kinases. The Rho kinases (≈160 kDa) include Rho kinase itself (ROKα or ROCK2) and the related p160ROCK (ROKβ or ROCK1, also known as Rho kinase). ROKα may stimulate LIM kinase to phosphorylate and inactivate cofilin.²⁷ Because cofilin promotes actin depolymerization,²⁸ the net effect of RhoA through this pathway is to promote formation of actin fibrils. RhoA activation of Rho kinase promotes phosphorylation of myosin light chains (MLCs) and inhibition of MLC phosphatase.⁴ This increase in MLC phosphorylation regulates cytoskeletal organization.

ET-1–induced hypertrophy is inhibited by the Rho kinase selective inhibitor Y27632,²⁹ and some dominant-negative mutants of Rho kinase inhibit PE-, ET-1–, or RhoA-induced hypertrophy,^{15 29} suggesting that Rho kinase is involved in the hypertrophic response. However, interpretation of some of these experiments may not be simple. For example, the Rho-binding domain of Rho kinase inhibits V14RhoA- or PE-induced myofibrillogenesis,¹⁵ but this may reflect general sequestration of activated RhoA rather than Rho kinase acting as a signaling intermediate. The kinase-dead form of Rho kinase should be a more specific inhibitor of Rho kinase signaling, but this did not inhibit PE-induced myofibrillogenesis and was less effective than the Rho kinase–binding domain in inhibiting myofibrillogenesis or ANF expression induced by activated RhoA.¹⁵ However, others showed that the kinase-dead Rho kinase inhibited ET-1–induced BNP expression.²⁹ Equally contradictory are the data indicating that N19RhoA,²⁰ but not the Rho kinase–binding domain,¹⁵ inhibits V12Ras-induced ANF expression. Thus, the role of Rho kinase in hypertrophy requires further clarification.

There is evidence that Rac1 participates in the hypertrophic response.^{22 23} In some cells, Rac1 activates PAKs,⁵ which may promote activation of ERKs^{30 31} or stimulate JNKs and p38-MAPKs.⁵ In cardiac myocytes, Rac1 cooperates with c-Raf to promote ERK activation and ANF expression (A.C. and P.H.S., unpublished data, 1999). Although Rho family proteins may promote JNK activation in cardiac myocytes, we have no evidence to suggest that they activate p38-MAPKs (A.C. and P.H.S., unpublished data).

As mentioned above, myocyte hypertrophy involves changes in cell architecture and cell-cell interactions. Activation of PAKs by Rac1 and Cdc42 regulates cell-cell interactions and cell architecture in other cells by modulating the actomyosin cytoskeleton and cell motility and migration.⁴ PAKs affect the phosphorylation state of cytoskeletal MLCs, although whether they promote or diminish phosphorylation is controversial. Thus, PAKs may phosphorylate and inactivate MLC kinase and diminish MLC phosphorylation (which should antagonize the effects of RhoA), but they have also been reported to promote MLC phosphorylation, potentially reinforcing the effects of RhoA.⁵ Whether the increase in MLC phosphorylation is mediated through MLC kinase is not clear, although PAKs may directly phosphorylate MLC.⁵ Additionally, PAKs may also phosphorylate LIM kinase,⁵ which may be expected to synergize with RhoA to promote cytoskeletal organization. To our knowledge, there is no direct evidence in cardiac myocytes to link activation of Rac1 to any of the downstream molecular-signaling events described here.

Note Added in Proof

The generation of a mouse cardiospecifically expressing a V12Rac1 transgene has been reported recently (Sussman MA, Welch S, Walker A, Klevitsky R, Hewett TE, Price RL, Schaefer E, Yager K. Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1. J Clin Invest. 2000;105:875–886). Two distinct phenotypes (a lethal neonatal dilated cardiomyopathy and a resolving transient cardiac hypertrophy in juveniles) were detected. Because PAK translocated from the cytoplasm to the cytoskeleton, the authors suggest that activation of Rac1 may induce reorganization of focal adhesions and the cytoskeleton, and this is an event common to both phenotypes.