Abstract
There is great interest in deciphering mechanisms of maladaptive remodeling in cardiac hypertrophy in the hope of affording clinical benefit. Potential targets of therapeutic intervention include the cytoplasmic phosphatase calcineurin and small guanosine triphosphate-binding proteins, such as Rac1 and RhoA, all of which have been implicated in maladaptive hypertrophy. However, little is known about the interaction—if any—between these important signaling molecules in hypertrophic heart disease. In this study, we examined the molecular interplay among these molecules, finding that Rho family guanosine triphosphatase signaling occurs either downstream of calcineurin or as a required, parallel pathway. It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition blocks hypertrophy, and we report here that “statin” therapy effectively suppresses small G protein activation and blunts hypertrophic growth in vitro and in vivo. Importantly, despite significant suppression of hypertrophy, clinical and hemodynamic markers remained compensated, suggesting that the hypertrophic growth induced by this pathway is not required to maintain circulatory performance.
Hypertrophic growth of the heart is an adaptive response to stress present in many forms of disease, including hypertension, ischemic disease, valvular disease, and inherited mutations of sarcomeric proteins.1In each of these conditions, hypertrophy is thought to play a critical role in enhancing cardiac output and normalizing wall stress, thereby decreasing the demand for oxygen. However, cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality, including arrhythmia, sudden cardiac death, and decompensation to heart failure.2,3As such, there is great interest in dissecting mechanisms underlying the maladaptive features of hypertrophy to provide specific targets for therapeutic intervention.
Among the major pathways leading to maladaptive hypertrophy is one mediated by the intracellular protein phosphatase calcineurin.4Activation of calcineurin is sufficient to induce severe hypertrophy that decompensates rapidly to failure and sudden death. Calcineurin catalytic activity can be suppressed pharmacologically or by interaction with one of several inhibitor proteins, including modulatory calcineurin-interacting protein (MCIP),5AKAP79, or Cain/Cabin.6,7Previous work has shown that suppression of the calcineurin pathway can be well tolerated.8As a result, there is great interest in elucidating upstream activators and downstream targets of calcineurin signaling in the hope of identifying novel targets of therapy.
The Rho family of guanosine triphosphatases (GTPases) integrates signals from numerous stimuli to regulate a host of downstream phenotypes, including cell growth and survival, cell morphology, and motility.9,10The Rho subfamily includes RhoA, Rac1, and Cdc42. These GTPases share some common upstream activators and downstream targets with calcineurin. For instance, Rac is activated by several established triggers of calcineurin, including α-adrenergic11or β-adrenergic12stimulation, angiotensin II (AngII),13and changes in intracellular calcium.14In T lymphocytes, calcineurin has been shown to act upstream of RhoA in the activation of serum response factor by muscarinic receptors.15Ras and mitogen-activated protein kinase signals facilitate nuclear factor of activated T cell (NFAT)-dependent transcription in many cell types.16,17Small GTPases participate in the regulation of ion channel activity in numerous cell types, including cardiac myocytes,18and cardiac-specific activation19or inhibition20of RhoA alters electrical conduction in the heart. Although it is well documented that signaling through small GTPases facilitates cardiac hypertrophy,8,21little is known about their possible interaction—if any—with calcineurin.
In this study, we set out to determine whether Rho family small GTPases are activated in calcineurin-dependent cardiac hypertrophy and, if so, whether they represent a viable target for therapeutic intervention. Initial experiments suggested activation of small GTPases by several hypertrophic agonists. Given the role of small GTPases as integrators of numerous hypertrophic triggers, we hypothesized that calcineurin activation would occur downstream. Rather, we observed that activation of small G protein signaling is situated downstream of (or in parallel with) calcineurin. Finally, in a pharmacologic study, we observed that 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibition suppressed small G protein activation and “statin” therapy blunted hypertrophic growth in vitro and in vivo. Finally, despite significant suppression of hypertrophy, clinical and hemodynamic markers remained compensated, suggesting that the hypertrophic growth induced by this pathway is not required to maintain circulatory performance.
EXPERIMENTAL PROCEDURES
Primary Culture of Neonatal Rat Ventricular Myocytes
Rat ventricular cardiomyocytes were isolated from the ventricles of 1- to 2-day-old Sprague-Dawley rat pups and plated as described22in medium containing 10% fetal calf serum to a density of 1,250 cells/mm2. Myocyte cultures obtained from this differential plating method contained less than 5% noncardiomyocytes as determined microscopically using a myocyte-specific α-actinin antibody (data not shown). Cells were transferred to serum-free medium containing the serum supplement Nutridoma (Roche, Indianapolis, IN) at a 0.5× concentration 24 hours before treatment or infection with adenovirus.
Adenoviral Infection of Cardiomyocytes
Experiments in which adenovirus expressing green fluorescent protein (AdGFP)-infected cells were simultaneously labeled with a fluorescent-tagged antibody recognizing α-actinin demonstrated that adenoviral particles at a multiplicity of infection (MOI) of 50 or higher infected 85 to 90% of cells (data not shown). Adenovirus containing dominant negative (N17Rac1, N19RhoA, and N17Cdc42) or constitutively active (V12Rac1, V14RhoA, and V12Cdc42) small G protein mutants, each coexpressing green fluorescent protein as a fusion protein, have been described.23Experiments were performed at MOI = 100 (≥ 95% infection).
Two-Dimensional Cell Surface Area
Myocytes were incubated with a fluorescent cell tracer (CellTracker, Molecular Probes, Carlsbad, CA) for 30 minutes and fixed in 4% paraformaldehyde. The two-dimensional cell surface area was measured using NIH Image software.
Immunostaining
Myocytes were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% bovine serum albumin, and incubated with antibodies to atrial natriuretic factor (ANF) or α-actinin and secondary antibodies conjugated to fluorescein isothiocyanate or Cy3 or with rhodamine-conjugated phalloidin.
Ribonucleic Acid Isolation and Analysis
Total ribonucleic acid (RNA) was prepared from neonatal cardiomyocytes using TriPure Isolation Reagent (Roche Molecular Biochemicals). ANF and brain-type natriuretic protein (BNP) messenger ribonucleic acid (mRNA) abundance was assessed by Northern dot blot analysis using radiolabeled probes derived from the rat ANF and mouse BNP complementary deoxyribonucleic acids (DNAs) and 2 μg of total RNA. Membranes were rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to normalize for differences in RNA loading. RNA expression levels were quantified using a Molecular Dynamics Storm PhosphorImager (Amersham Biosciences, Piscataway, NJ).
Guanosine Triphosphate-Binding Activities
To begin to quantify small G protein activation, we measured the guanosine triphosphate (GTP)-bound, active fraction of RhoA and Rac1 in membrane fractions as described.24Briefly, 20 μg of membrane protein was incubated for 30 minutes at 37°C in an incubation buffer containing [35S]GTPγS (20 nmol/L) and 0.002 GTP (solution in mmol/L unless otherwise indicated), 5.0 MgCl2, 0.1 egtazic acid (EGTA), 50 NaCl, 4.0 creatinine phosphate, phosphocreatine kinase (5 units), 0.1 adenosine triphosphate, 1.0 dithiothreitol, leupeptin (100 μg/mL), aprotinin (50 μg/mL), and 2.0 phenylmethanesulfonyl fluoride (PMSF). The assay was terminated by the addition of excess unlabeled GTPγS (100 μmol/L), and samples were resuspended in 100 μL of immunoprecipitation buffer containing Triton-X (1%), sodium dodecyl sulfate (SDS) (0.1%), 150 NaCl, 5.0 ethylenediaminetetraacetic acid, 25 Tris-HCl (pH 7.4), leupeptin (10 μg/mL), aprotinin (10 μg/mL), and 2.0 PMSF. Rac1 or RhoA antiserum was added to the mixture at a final dilution of 1:75, and samples were allowed to incubate for 4 hours with gentle mixing. Antibody-G protein complexes were incubated with 50 μL protein A-Sepharose for 2 hours, and the immunoprecipitate was collected by centrifugation at 12,000g for 10 minutes. The recovered pellets were then washed four times in a wash buffer containing 50 HEPES (pH 7.4), 0.1 NaF, 50 sodium phosphate, 100 NaCl, Triton X-100 (1%), and SDS (0.1%). The final pellet was then counted in a liquid scintillation counter, with nonspecific activity determined in the presence of excess unlabeled GTPγS (100 μmol/L).
Pressure-Overload Hypertrophy Model
Male C57BL6 mice (6-8 weeks old) were subjected to pressure overload by thoracic aortic banding (TAB).25,26At the time of tissue harvesting, the integrity of the surgical band was confirmed by inspection of bilateral carotid arteries. In occasional animals, proximal aortic pressures were measured in anesthetized mice as described.25
Statin Treatment
Mice were randomized to TAB or sham operation. On the first postoperative day, animals were randomized a second time to simvastatin (2 mg/kg subcutaneously twice daily) or vehicle injection. Dose-ranging (simvastatin 1 mg/kg twice daily) and control (2 mg/kg/d simvastatin prodrug or vehicle injection) experiments were performed.
Echocardiography
Transthoracic echocardiograms were recorded in conscious sedated mice as described.25Left ventricular internal diameters and wall thicknesses were measured (at least three cardiac cycles) at end-systole and end-diastole from two-dimensionally targeted M-mode cross-sectional views at the level of the chordae tendineae. Heart rate was determined from mitral inflow Doppler envelopes.
Reagents
Reagents were procured as follows: AngII (Sigma, St. Louis, MO), NTDP (Sigma), A23187 (Sigma), Losartan (Merck, Whitehorse Station, NJ), PD123319 (Sigma), cyclosporin A (Sigma), Neonatal Cardiomyocyte Isolation System (Worthington Biochemical, Lakewood, NJ), atorvastatin (Pfizer), simvastatin (Merck), mevalonate (Sigma), geranylgeranyl transferase inhibitor (GGTI)-286 (Sigma), α-hydroxyfarnesylphosphonic acid (Sigma), 35S-GTPγS (Amersham), and Rac1/RhoA antibodies (Santa Cruz).
Statistics
Data are expressed as mean ± standard error of measurement. Statistical significance was analyzed with a Student's unpaired t-test or one-way analysis of variance followed by Bonferroni's method for post hoc pairwise multiple comparisons. A p value < .05 was considered significant.
RESULTS
To examine the interaction of small G proteins and calcineurin signaling during hypertrophy, we used a well-established model of rat neonatal cardiomyocytes in culture. AngII treatment induced significant increases in cell size via a calcineurin-dependent mechanism (Figure W1). AngII-induced hypertrophy was mediated by angiotensin receptor type 1 (AT1) receptors because losartan blocked cell growth and PD123319, a specific blocker of the AT2 receptor, had no effect (see Figure W1). Forced overexpression of a constitutively active mutant of calcineurin (CnA) also induced significant hypertrophic growth (see Figure W1).
Post-translational isoprenylation is a required processing step in the activation of small G proteins, and depletion of intracellular isoprenoid intermediates blocks GTPase activation.27Cotreatment of cells with an HMG CoA reductase inhibitor (atorvastatin) inhibited AngII-mediated hypertrophy in a dose-dependent manner (Figure 1A). A similar dose response was obtained with simvastatin (data not shown). To determine the specificity of the effects of statins, cells were exposed to an inactive, prodrug isoform of simvastatin, which had no effect (Figure 1B). Statin blockade of hypertrophy was specific to reductase enzymatic activity because exposure to mevalonate, the immediate downstream product of HMG CoA reductase, restored the growth response (Figure 1C).
Isoprenylation of Rho and Ras preferentially occurs as geranylgeranylation and farnesylation, respectively.27To obtain additional evidence for involvement of small G proteins in calcineurin-dependent hypertrophy, cells were exposed to an inhibitor of geranylgeranylation (GGTI-286), which prevented AngII-induced hypertrophy (Figure 1D). In contrast, inhibition of farnesylation with α-hydroxyfarnesylphosphonic acid had no effect. Together, these data provide evidence of Rho family involvement in AngII-induced hypertrophy and argue against the involvement of Ras.
To test the role of specific small G proteins, cardiomyocytes were exposed to AngII and simultaneously infected with adenovirus-expressing dominant negative mutants of Rho family GTPases Rac1 (N17Rac1), RhoA (N19RhoA), and Cdc42 (N17Cdc42). These dominant negative mutants are specific and highly efficacious inhibitors of individual GTPases; consequently, they are widely used. Suppression of Rac1 or RhoA activity using these reagents prevented AngII-dependent increases in cell surface area (Figure 2A) and sarcomeric organization (Figure 2B), a hallmark of hypertrophy. Overexpression of dominant negative Cdc42, another Rho family GTPase, had no effect, demonstrating the specificity of the reaction to RhoA and Rac1.
To begin to quantify activation of RhoA and Rac1, we measured the GTP-bound (active) fraction of RhoA and Rac1 in AngII-treated and control cardiomyocytes by immunoprecipitating [35S]GTPγS-labeled RhoA and Rac1. In these experiments, we found that GTP-bound RhoA increased rapidly following treatment with AngII (Figure 3A). This increase was sustained for at least 30 minutes and was blocked by atorvastatin. The abundance of GTP-bound, active Rac1 also increased in response to AngII treatment, although with a temporal delay compared with RhoA (Figure 3B).
Small G Protein Activation Is Situated Downstream of Calcineurin
Small G proteins function as integrators of diverse signals in numerous cell types.28Thus, we tested whether small G protein signaling acts upstream or downstream of calcineurin during the hypertrophic response of cardiomyocytes. Myocytes were infected with adenovirus expressing a CnA and then treated with atorvastatin or vehicle. Strikingly, atorvastatin abolished the calcineurin-dependent hypertrophic growth response measured as cell size (Figure 4A) or sarcomere organization (Figure 4B). Again, the effects of atorvastatin were found to be dependent on HMG CoA enzymatic activity because cotreatment with mevalonate restored the hypertrophic response. Dominant negative mutants of RhoA and Rac1 blocked the hypertrophic growth response of myocytes to activated calcineurin, whereas dominant negative Cdc42 did not (Figure 4, C and D).
To examine the effect of calcineurin signaling on GTPase activation, we measured the GTP-bound fraction of RhoA and Rac1 in myocytes infected with adenovirus-expressing activated calcineurin. The abundances of GTP-bound, active RhoA and Rac1 were higher in cardiomyocytes infected with activated calcineurin compared with control cells (Figure 5). Atorvastatin inhibited the increase in activated RhoA and Rac1 levels, whereas cotreatment with mevalonate restored those levels. Taken together, these findings suggest that small G protein activation occurs downstream of calcineurin signaling.
To test whether calcineurin is activated downstream of small G protein-induced hypertrophy and thus contributes to the hypertrophic response, cultured myocytes were infected with adenovirus-expressing constitutively active mutants of Rac1 (V12Rac1), RhoA (V14RhoA), and Cdc42 (V12Cdc42). Calcineurin was suppressed either pharmacologically or by coinfection with adenovirus-expressing modulatory calcineurin-interacting protein 1 (MCIP1), an endogenous inhibitor of calcineurin activity.29As expected,8overexpression of activated Rac1 and RhoA significantly increased cell surface area compared with vehicle-treated and AdGFP-infected controls (Figure 6A). Activated Rac1 and RhoA also promoted sarcomeric organization, whereas activated Cdc42 and AdGFP did not (see Figures 4D and 6B). Neither cyclosporinA (CsA) nor MCIP had any effect on cell surface area or sarcomeric organization induced by Rac1 or RhoA (see Figure 6, A and B), whereas these treatments were effective in blocking cell growth triggered by AngII or CnA (data not shown). These data, then, suggest that downstream calcineurin activation is not required for Rac1- or RhoA-induced cardiomyocyte hypertrophy in vitro.
ANF Expression and Secretion
One hallmark of pathologic hypertrophy is the activation of a pattern of gene expression that mimics that observed during fetal development (fetal gene program). Among these genes are those coding for ANF, BNP, and β-myosin heavy chain. To determine the pattern of ANF gene activation elicited by the calcineurin-small GTPase pathway, steady-state transcript levels were measured by RNA dot blot hybridization. Significant increases in ANF transcript were observed following exposure to activated CnA (Figure 7) or Ang II (data not shown). Cotreatment with atorvastatin or mevalonate (or both) did not significantly inhibit this increase in mRNA abundance. This suggests that statin treatment inhibits calcineurin-mediated hypertrophic growth through mechanisms other than altering the transcriptional response of fetal genes to calcineurin activity.
The expression of ANF and BNP is elevated in both failing and hypertrophied ventricles. Although indicative that the heart is under stress, these molecules have antihypertrophic properties because mice lacking the gene encoding guanylyl cyclase A, a common receptor for ANF and BNP, have marked hypertrophy and fibrosis.30,31ANF gene expression was neither increased nor inhibited by statin treatment (see Figure 7). As the role of small G proteins in membrane trafficking is well established, we examined whether trafficking of the ANF protein was altered. As expected, both AngII and adenoviral expression of activated calcineurin catalytic subunit (AdCnA) induced dramatic accumulation of ANF protein in a perinuclear pattern (Figure 8). Interestingly, this accumulation was not inhibited when AngII and AdCnA-treated cells were coinfected with dominant negative small G proteins or atorvastatin. These data, then, suggest that hypertrophy-associated trafficking of ANF peptide may not involve small G proteins, which is consistent with previous data implicating microtubule-associated granules in the regulated exocytosis of ANF.32
In Vivo Pressure Overload
An expanding body of literature points to inhibition of hypertrophic signal transduction as an attractive direction for future research; in numerous animal models, suppression of hypertrophic growth is well tolerated, with preservation of ventricular size and performance, despite persistence of the inciting stimulus.8Given this, we set out to determine whether HMG CoA reductase inhibitors—widely prescribed, well-tolerated drugs that have been proposed as a treatment strategy in cardiac hypertrophy and failure33—would suppress hypertrophic growth in a model of clinically relevant load-induced hypertrophy in vivo. To test this, mice were subjected to TAB and randomized on the first postoperative day to treatment with simvastatin (2 mg/kg subcutaneously twice daily) or vehicle injection. At 3 weeks, when the hypertrophic response reaches steady state,25animals were sacrificed and subjected to necropsy analysis. As expected, TAB mice randomized to vehicle injections exhibited significant increases in heart and left ventricular mass (Figure 9). Statin-treated TAB mice manifested significantly (p < .05) smaller increases in heart mass, suggesting a blunted hypertrophic response. Inactive prodrug had no effect (p = not significant) on the cardiac growth response to TAB (n = 5; data not shown).
Some evidence implicates RhoA and Rac1 in “maladaptive” hypertrophy,34-36which led us to hypothesize that prevention of hypertrophy with statin therapy will be well tolerated. Consistent with this hypothesis, animals subjected to TAB but in whom hypertrophy was blunted by statin therapy were clinically normal, without evidence of cardiovascular compromise, such as lethargy, weight gain, or edema. Cardiac function analyzed by echocardiography revealed preservation of ventricular size and systolic performance in TAB mice treated with simvastatin (Table 1). These findings reveal that statin inhibition of hypertrophy can be well tolerated under conditions of moderate stress, suggesting that the hypertrophic growth induced by this pathway is not required to maintain circulatory performance.
DISCUSSION
In this report, we demonstrate for the first time that calcineurin-mediated hypertrophy is dependent on activation of the Rho family of small G proteins. Hypertrophic signals trigger GTPase activation, and inhibition of GTPase activity is sufficient to prevent cell growth. Extending these findings to a model of load-induced hypertrophy that mimics clinical forms of disease, we demonstrate that HMG CoA reductase inhibition blunts the pathologic growth of the heart. Finally, despite attenuation of hypertrophic growth, cardiac size and performance were preserved in the face of persistent afterload stress.
We amassed several lines of evidence implicating small G protein activation in calcineurin-dependent hypertrophy. First, HMG CoA reductase inhibitors, drugs that are capable of inhibiting small G proteins, block hypertrophy triggered by several agonists. Second, dominant negative mutants of RhoA and Rac1 block hypertrophy triggered by AngII and by activated calcineurin. Third, the abundance of the active fractions24of RhoA and Rac1 (indirect measures of their activation) was increased by AngII and activated calcineurin. Fourth, pressure-overload stress, a growth trigger known to require calcineurin, is attenuated by statins. Fifth, the observation that hypertrophy induced by constitutively active small G proteins is not inhibited by calcineurin blockers situates these molecules downstream of or parallel with calcineurin.
The ability of calcineurin signaling to promote the hypertrophic growth of cardiac myocytes is well established.8Whereas most studies have focused on the transcription factors NFAT and myocyte enhancer factor 2 as the major downstream mediators of calcineurin-dependent gene activation, other mechanisms through which calcineurin may be acting have been described, including interactions with membrane proteins such as ion channels.37Prior to this work, however, crosstalk between calcineurin and signaling via small G proteins had not been examined in cardiac myocytes.
Small G proteins of the Rho family are a point of convergence of several pathways important in hypertrophy, including those stimulated by AngII, mechanical stress, and growth factors.38-40Prior to this study, it was not known whether small G proteins acted upstream or downstream of Ca2+ /calcineurin signaling during the hypertrophic response. In vitro studies using cardiac myocytes suggest that calcineurin is a downstream target of Ras.41Numerous studies have pointed to the Rho family as a mediator of AngII-induced hypertrophy in vitro.13,40GATA4, a transcription factor that partners with calcineurin-activated NFAT to activate transcription of hypertrophic genes, including ANF, is a downstream effector of RhoA in cardiomyocytes.42Based on these data, we originally hypothesized that small G protein signaling would act upstream of calcineurin, possibly functioning to integrate diverse signals by coincidence detection. We found, however, that inhibition of the small G proteins Rac1 and RhoA blocked myocyte hypertrophy induced directly by activated calcineurin and that hypertrophy in response to direct activation of small G proteins is insensitive to calcineurin suppression. These results suggest that activation of Rho family GTPases is situated downstream of calcineurin or that activation of Rho signaling is a necessary and parallel pathway for the development of calcineurin-dependent hypertrophy.
We observed partial redundancy among Rho family GTPases; dominant negative mutants of either RhoA or Rac1 were sufficient to block hypertrophy, whereas Cdc42 was not. This is consistent with the known crosstalk between Rac and Rho.10Cardiac-specific overexpression of either Ras43or Rac144in transgenic mice is sufficient to trigger cardiac hypertrophy and cardiomyopathy. Conversely, overexpression of RhoA in mouse myocardium does not lead to hypertrophy but rather targets the L-type Ca2+ channel18and induces atrial and electrophysiologic abnormalities.19
HMG CoA reductase inhibition has been shown to prevent hypertrophy induced by a variety of stimuli.8For example, Laufs and colleagues tested the effects of statin treatment on neonatal cardiomyocytes exposed to high-dose AngII (10 μM), finding that simvastatin abolished AngII-induced increases in ANF expression.45In this study, we extend these observations by demonstrating that statin therapy can block hypertrophy of cardiomyocytes even in the presence of activated calcineurin. Thus, inhibition of small G proteins can override a persistent calcineurin signal. Furthermore, statin-mediated blockade of hypertrophy is well tolerated in vivo. This latter observation lends credence to the notion that small GTPase-dependent hypertrophy is maladaptive and supports further work to explore these drugs as an antihypertrophic treatment strategy. Indeed, recent studies have demonstrated that therapeutic targeting of hypertension-induced hypertrophy is a viable strategy that confers long-term mortality benefit.46,47
Although this study increases our understanding of the molecular interactions between calcineurin and GTPases, numerous questions remain. For example, Rac1 and RhoA activation increases reactive oxygen species (ROS) levels, which leads to hypertrophy in the heart34and vascular smooth muscle.24Further work will be required to elucidate the contribution of ROS in calcineurin-triggered small G protein signaling. Whether downstream Rho signaling occurs in series with calcineurin or as a required cascade active in parallel is unknown. Finally, the long-term tolerability of statin therapy at doses used to prevent hypertrophy (approximately three to four times greater than doses commonly used in lipid-lowering therapy) remains to be determined. Together, however, these studies elucidate the molecular interplay between calcineurin and small G proteins and raise the prospect of therapeutic intervention in hypertrophy by well-tolerated drugs such as statins.
ACKNOWLEDGMENTS
We thank Dr. Jeffrey Molkentin for providing us with adenovirus-expressing activated calcineurin and Tomasa Barrientos and Eric Olson for their valuable assistance.