pmc.ncbi.nlm.nih.gov

Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice

Abstract

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of β-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat α-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dt_max) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dt_min, was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, β-adrenergic receptor (β-AR) density, and β-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-β is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-β/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting β-AR signaling capacity.

Akt is involved in receptor-mediated signaling, acting as a critical mediator of cell size and survival in numerous cell types (reviewed in ref. 1). Akt family proteins contain a central kinase domain with specificity for serine or threonine residues in substrate proteins. In addition, the amino terminus of Akt includes a pleckstrin homology (PH) domain, which mediates lipid–protein and/or protein–protein interaction (2). The Akt carboxyl terminus includes a hydrophobic and proline-rich domain. Diverse arrays of physiological stimuli can induce Akt kinase activity, primarily through phosphatidylinositol 3-kinase (PI3K). PI3K can be activated through ligation of transmembrane receptors, which either possess intrinsic tyrosine kinase activity (e.g., insulin-like growth factor-1 or insulin receptor) or are indirectly coupled to tyrosine kinases (e.g., IL6 family receptors) or to seven transmembrane G protein-coupled receptors [e.g., β-adrenergic receptor (β-AR)]. Once localized to the plasma membrane, PI3Ks catalyze the transfer of phosphate from ATP to the D-3 position of the inositol ring of membrane-localized phosphoinositides. PI3Ks principally generate phosphatidylinositol 3,4 bisphosphate (PI3,4P) and phosphatidylinositol 3,4,5 triphosphate (PI3,4,5P). Once generated, these lipids then function as signaling intermediates that regulate down-stream signal transduction cascades. It was found that PI3K activity is required for the growth factor-dependent survival of a wide variety of cell types. PI3K-generated phospholipids regulate Akt activity by direct binding of phosphoinositides to the PH domain, translocating Akt from the cytoplasm to the inner surface of the plasma membrane and rendering Akt available to be phosphorylated by PDK1 at Thr-308 and PDK2 at Ser-473 (1).

In cardiomyocytes, Akt is activated after gp130 stimulation and has both a hypertrophic (3) and an antiapoptotic effect (4). β-Adrenergic signaling also activates Akt. Both PI3K and calcium-calmodulin kinase are responsible for Akt activation after β₁- or β₂-adrenergic stimulation (5, 6). Transcriptional activation of the atrial natriuretic factor promoter and cell hypertrophy following β₁-AR stimulation were proved to depend upon Akt activation (5). In fact, it was demonstrated that glycogen synthase kinase3 (GSK3)-β regulates subcellular localization of the zinc finger transcription factor GATA4 and that β₁AR stimulation induces GSK3-β phosphorylation/inactivation followed by nuclear accumulation of GATA4 (7). It was also demonstrated that Akt is responsible for the antiapoptotic effect of β₂AR stimulation (8). In myocardial gene transfer studies, it was shown that a constitutively active mutant of Akt reduces infarct size and apoptosis after ischemia-reperfusion injury (4). Moreover, it was shown that viral-mediated Akt overexpression restored ventricular wall thickening and maximal rate of left ventricular diastolic pressure rise and fall (+dP/dt and −dP/dt; ref. 9). This study also suggested that Akt enhances cardiac metabolism by increasing glucose uptake and enhancing Glut-4 expression (9). Indirect evidence of the role of Akt in mediating survival and apoptosis in cardiomyocytes was provided by a study in which phosphatase and tensin homolog (PTEN) was overexpressed, causing apoptosis together with dephosphorylation of Akt (10). Furthermore, a transgenic mouse approach has been used to overexpress a constitutively active PI3K in cardiomyocytes, which generated concentric hypertrophy (11). Similarly, overexpression of an active form of GSK3-β attenuated isoproterenol (ISO)-induced hypertrophy in vivo (12).

In this article, we describe the characterization of a transgenic line of mice overexpressing a constitutively active form of Akt, the E40K mutant, in the heart. We show here that Akt E40K induces a striking increase of myocardial mass and, unexpectedly, a remarkable increase in cardiac contractility.

Materials and Methods

Generation of Transgenic Animals.

An insert of the E40K mutant of Akt was blunted and cloned in the unique blunted SalI site of α-myosin heavy chain (α-MHC)-SV40-INS-PolyA (13). The α-MHC promoter region was a gift of J. Robbins (Children's Hospital, Cincinnati). E40K mutant is a constitutively active form of Akt which can localize in many subcellular locations, not only to the plasma membrane (2). Numerous lines of transgenic mice were obtained. Lines were selected for expression of Akt, and only two lines highly expressing Akt were carried on. All of the experiments were done by using line 6445.

Western Blots.

Western blots were carried out on myocardial extracts. Thirty micrograms of proteins were loaded on gels and blots were analyzed with antibodies against Akt and phosphorylated Akt (phospho Ser-473 or Thr-308), which were used according to manufacturer's instructions (Cell Signaling, Beverly, MA). Similarly, antibodies for mitogen-activated protein kinase (MAPK) p42/44, their phosphorylated forms, and GSK3-β (phospho Ser-9) were obtained from Cell Signaling and used according to instructions.

Adenoviruses and Cardiomyocyte Cell Culture.

Two Akt adenoviruses were used in this study: one expressing a dominant-negative mutant, Akt T308A/S473A, and another expressing a constitutively active mutant, Akt-Myr (2). Rat neonatal cardiomyocytes were prepared as described (14).

Histology and Electron Microscopy.

Masson trichrome and wheat germ hemagglutinin staining were performed essentially as described (15, 16) from sections of hearts fixed through retrograde aortic perfusion.

Similarly, electron microscopic analysis was performed as described (13). Briefly, ultrastructural analysis of morphological changes in transgenic mice was performed after fixing the heart in conventional fixing solutions [4% (vol/vol) paraformaldehyde and 1% (vol/vol) glutaraldehyde in 0.1M phosphate buffer]. Samples were processed (postfixation and dehydration) for embedding in epoxy resin. Ultra-thin sections, stained with uranyl acetate and lead hydroxide, were studied by using a CM-12 Philips electron microscope.

Echocardiography.

By using methods somewhat modified from those described (17), mice were anesthetized with Avertin (tribromoethylene, 2.5%, 15–17 μl/g) given i.p., and transthoracic echocardiography was performed by using an Agilent Technologies Sonos-5500 echocardiograph with a 15-MHz linear transducer.

Because of the high heart rates, which cause fusion of the A and E waves, diastolic function was not evaluated by Doppler.

Hemodynamic Studies.

Mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg, i.p.), ventilated, and a 1.4 French Millar catheter-tip micromanometer catheter was inserted through the right carotid artery into the left ventricle, where pressure was recorded. True zero was obtained in saline at the end of each experiment, and any offset was corrected. This anesthetic results in deeper anesthesia, which is required for hemodynamic studies, and causes slowing of the heart rate. Data were reported before bilateral vagotomy (Table 1) and after vagotomy, which reduces heart-rate variability; under the later condition, heart rates were higher and nearly identical. After the vagotomy, basal pressures were again measured, and β-adrenergic responsiveness was assessed with graded doses of dobutamine, as described (18).

Table 1.

Hemodynamic data prior to vagotomy

	Type	HR, bpm	Max LVP, mmHg	EDP, mmtHg	Max dP/dt, mmHg/sec	Min dP/dt, mmHg/sec	tau, msec
Mean	TG (n = 14)	353	102	5.0	10,290	−6553	13.4
SD		± 58	± 18	± 3.7	± 1,614	± 1883	± 2.8
Mean	WT (n = 9)	311	93	7.6	6,410	−5796	12.6
SD		± 61	± 8	± 4.7	± 1,134	± 1472	± 1.8
P		n.s.	n.s.	n.s.	<0.001	n.s.	n.s.

β-AR Binding, Affinity, and Adenylyl Cyclase Assay.

Assays were performed as described (16). Briefly, myocardial membranes were prepared by homogenization of excised hearts in ice-cold lysis buffer (50 mmol/liter Hepes, pH 7.3/150 mmol/liter KCl/5 mmol/liter EDTA). Final membrane preparations were resuspended at a concentration of 2–3 mg/ml in ice-cold β-AR binding buffer (75 mmol/liter Tris⋅Cl, pH 7.4/12.5 mmol/liter MgCl₂/2 mmol/liter EDTA) and binding was performed with the β-AR ligand [¹²⁵I]cyanopindolol, as described (16). All assays were performed in triplicate, and receptor density (femtomoles) was normalized to milligrams of membrane protein.

Adenylyl Cyclase Activity.

Crude myocardial membranes were prepared as described above. Membranes (20–30 μg of protein) were incubated for 15 min at 37°C with [α-³²P]ATP under basal conditions or in the presence of either 0.1 mmol/liter ISO or 10 mmol/liter NaF, and cAMP was quantitated by standard methods, as described (16).

Results

Cardiac-Specific Akt Overexpression Affects the GSK3-β/GATA4 Axis but Not p42/44 MAPK Activation.

Overexpression of the E40K Akt mutant linked to the α-MHC promoter induces robust cardiac selective expression. In fact, Western blotting of myocardial extracts shows a remarkable increase of Akt levels in transgenic but not control animals (Fig. 1A). Such expression is sustained, because mice at 6 months of age showed similar levels of Akt proteins to those at 8 to 12 weeks (data not shown). Akt phosphorylation, an index of its activation, also is increased in transgenic mice compared with controls, where it is almost not detectable (Fig. 1A).

Fig 1. — (A) Western blotting of myocardial cell extracts from three control (C1, C2, C3) and three transgenic (T1, T2, T3) animals. Upper lane, tissue extracts were blotted with an antibody recognizing both phosphorylated and unphosphorylated forms of Akt; lower lane, tissue extracts were blotted with an antibody against only the phosphorylated form of Akt. (B) Western blotting with an antibody against phospho-GSK3-β of myocardial cell extracts from Akt-transgenic and control mice. (*Left*) Responses are also shown in extracts of primary rat neonatal myocardial cells unstimulated and stimulated with ISO or infected with adenoviruses carrying a dominant-negative (Akt-dn) or a constitutively active (Akt-ca) form of Akt, respectively. (C) Immunohistochemistry using an anti-GATA4 antiserum in myocardial tissue from Akt transgenic or control mice. GATA4 is accumulated in the nucleus in the myocardium of Akt transgenic but not in control mice, where the coloration is more diffuse. (D) Western blotting with antibodies recognizing total MAPK p42/44 (upper lane) or phospho MAPK p42/44 (lower lane) in control (C1, C2, C3) vs. transgenic mice (T1, T2, T3).

It has been clearly shown that a critical downstream target of Akt is GSK-3β, which Akt inactivates by phosphorylation. Thus, we determined whether GSK-3β is phosphorylated in myocardial extracts from E40K mice as compared with control animals. Extracts from cardiomyocytes stimulated with the β-AR agonist ISO or infected with a constitutively active mutant of Akt or a dominant-negative form of Akt were included. We found that extracts obtained from transgenic mice and from cardiomyocytes infected in vitro with constitutively active Akt adenovirus or challenged with ISO demonstrated the presence of phosphorylated GSK3-β (Fig. 1B). Moreover, after phosphorylation of GSK3-β, GATA4 accumulates in the nucleus. In fact, we see nuclear localization of GATA4 in the myocardium of transgenic but not of control animals (Fig. 1C).

As with Akt, phosphorylation of MAPK is a hallmark of its activation. Western blotting with protein extracts from transgenic and control mice incubated with anti-phospho-MAPK p42/44 antibodies did not show phosphorylation (Fig. 1D).

E40K Expression Increases Cardiomyocyte Size and Induces Cardiac Hypertrophy.

Different lines of evidence, including echocardiographic analysis, postmortem weight measurements, and tissue histochemistry concurred in demonstrating the hypertrophic effects of Akt on cardiomyocytes in this transgenic mouse model.

Echocardiographic analysis was performed on mice of 8 to 11 weeks of age. The left ventricular (LV) end-diastolic diameter was not significantly different from wild-type mice, but the end-diastolic posterior and septal wall thicknesses were increased significantly by 32%, indicating concentric LV hypertrophy (Table 2). There were no significant differences between the two groups in echocardiographic measures of systolic function (% FS and VCF, which were normal); however, the mean heart rate was significantly lower in transgenic mice.

Table 2.

Echocardiographic data and postmortem heart weight data

	Type	BW	HR, bpm	SEPD, mm	PWD, mm	EDD, mm	FS %	VCF, circ/s	LV/BW	H/BW
Mean	TG (n = 14)	25.07	394	0.85	0.87	3.53	43.1	8.2	0.45	0.68
SD		± 3.92	± 54	± 0.17	± 0.19	± 0.46	± 11.1	± 2.4	± 0.13	0.134
Mean	WT (n = 9)	23.94	467	0.67	0.66	3.53	38.7	7.6	0.366	0.492
SD		± 2.75	± 83	± 0.05	± 0.04	± 0.27	± 8.5	± 2.1	± 0.05	0.070
P		n.s.	<0.01	<0.005	<0.005	n.s.	n.s.	n.s.	<0.01	<0.002

Postmortem analysis included weights of the total heart, left and right ventricles, left and right atria, LV weight to body weight (BW) and heart weight (HW) to BW ratios in transgenic and control animals. Weights of all of the cardiac chambers were significantly increased in transgenic animals compared with controls (data not shown); the HW/BW and LV/BW were also significantly increased (Table 2).

A third line of evidence demonstrating the hypertrophic effect of Akt on cardiomyocytes comes from histochemical comparison of the LV tissue of transgenic and control mice. Sections of hearts showed a remarkable increase of myocyte cell size in transgenic vs. control mice, using both Masson trichrome and wheat-germ agglutinin fluorescein-conjugated staining (Fig. 2 A, B, C, and D, respectively), as reported (16). The amount of collagen did not seem to be increased.

Fig 2. — (A and B) Masson trichrome staining of longitudinal sections of the myocardium from wild-type (A) and transgenic mice (B). (C and D) Staining with FITC-labeled wheat germ hemagglutinin of crosssectional sections of myocardium from Akt control (C) and transgenic animals (D).

Electron microscopy performed on the hearts of two transgenic and two control mice showed increased myofibrillar size in the transgenic but not in the control hearts, along with increased cell size (Fig. 3).

Fig 3. — Electron microscopy analysis of myocardial sections from control (A) or E40K transgenic mice (B). An increase in myofibrillar size is evident in transgenic mice.

Invasive Hemodynamic Analysis of Wild-Type and E40K Akt Transgenic Mice.

Measurements of LV intracavitary hemodynamic parameters, including peak LV pressure, LV end-diastolic pressure (EDP), LV dP/dt_max (an index of myocardial contractility), and LV dP/dt_min and tau (the time constant of LV relaxation), both indices of relaxation, were studied in wild-type and E40K Akt transgenic mice. After anesthesia and LV catheterization and before vagotomy, the heart rates were not significantly different, and the LV dP/dt_max in transgenic mice was markedly increased (by 67%) compared with that in wild-type mice (Table 1). Other hemodynamic variables were not significantly different. After vagotomy, the heart rates increased substantially in both groups but were not significantly different; LV dP/dt_max in transgenic mice remained substantially higher than in the wild-type mice (Fig. 4). Other hemodynamic variables including LV EDP and indices of LV relaxation were not significantly different from values in wild-type mice (Fig. 4). The LV dP/dt_max at the basal level and with initial doses of dobutamine was significantly higher in transgenic mice, whereas the response at the maximum dose was not significantly different between the two groups (Fig. 4). These results indicate that E40K overexpression increases basal myocardial contractility and initial dobutamine responses without affecting other hemodynamic variables, including LV dP/dt_min. However, the LV dP/dt_min response was significantly reduced during graded dobutamine infusion in transgenic vs. control animals at doses higher than 0.75 μg. Thus, diastolic function expressed as LV relaxation is altered in this model during stress.

Fig 4. — Hemodynamic analysis of cardiac function in Akt transgenic vs. control mice under resting conditions and with graded dobutamine infusion. (*Top*) Heart rate vs. dobutamine dosage in transgenic (•) vs. control (□) mice before vagotomy (control), after vagotomy (basal), and at different dobutamine dosages, as indicated in the figure. (*Top Middle*) Maximal LV pressure vs. dobutamine dosage in transgenic (•) vs. control (□) mice. (*Bottom Middle*) LV dP/dt_max in transgenic (•) vs. control mice (□). (*Bottom*) LV dP/dt_min in transgenic (•) vs. control mice (□). †, P< 0.05.

β-AR Function in Akt Transgenic Mice.

Data so far shown strongly suggest that Akt is a downstream mediator of the biological effects of β-AR. In fact, in conditions of β-AR overstimulation, such as during heart failure, kinase-dependent counterregulatory mechanisms activated by β-AR themselves take place, decreasing β-AR signaling. The last phenomenon is caused at least in part by an increased level of β-ARK protein, a G protein regulating kinase that inhibits β-AR signaling and affects also the affinity of β-AR for its ligands, as well as by decreased levels of β-AR on the cell membrane. ISO-induced cAMP generation, β-AR density and affinity were, therefore, evaluated in transgenic vs. control heart membranes. Results of these experiments show that all three variables were similar in transgenic compared with nontransgenic mice (Fig. 5). Moreover, levels of β-AR kinase, determined by Western blotting, were similar in transgenic vs. control animals (not shown). Thus, although a target of β-AR signaling, Akt activation itself does not seem to affect β-AR activation status.

Fig 5. — (A) ISO-induced cAMP generation in control vs. transgenic mice. Base is represented in white bars, ISO in black bars. (B) β-AR density (fmol/mg of membranes) in control vs. transgenic myocardium. (C) β-AR affinity, expressed as % of high-affinity agonist binding. The transgenic overexpression of AKT in the heart did not alter β-AR signaling nor affect β-AR receptor density or the ability of the receptor to couple to Gs.

Discussion

Data presented here indicate that Akt exerts a dual function in vivo on myocardium by increasing both cardiomyocyte size and contractility. In vivo, a slower heart rate also was characteristic of the phenotype in the Akt transgenic mice under the relatively light anesthesia used for echocardiography (Avertin), because under identical anesthesia the heart rate was substantially higher in the wild-type littermates; this slower heart rate in the transgenic group probably limited the increase in LV function assessed by the fractional shortening and VCF as a consequence of the negative inotropic effect of slower heart rate (negative force-frequency effect). However, myocardial contractility was substantially enhanced on hemodynamic study by using the widely accepted measure of LVdP/dt_max when the heart rates in transgenic and wild-type mice were not different. Recent reports using isolated cultured cardiomyocytes have identified Akt as both a target and a mediator of the downstream effects of β₁- and β₂-AR signaling. Akt is in fact responsible for most of the hypertrophic effects induced by both β₁- and β₂-AR activation on cardiomyocytes in vitro (5–7). Our in vivo data are in line with such a hypothesis, because Akt overexpression induced a remarkable increase in cardiomyocyte cell size in the myocardium of transgenic animals. Our results suggest that the pathway activated by the Akt responsible for its hypertrophic effect in vivo is the GSK3-β/GATA4 pathway, as already described in isolated cells (5, 7). In fact, β-AR sequentially activates Akt, which then phosphorylates GSK3-β, which is bound to GATA4 in the cytoplasm and in the nucleus. Once phosphorylated, GSK3-β releases GATA4, which then accumulates in the nucleus and activates transcription (5, 7). In myocardial extracts from transgenic animals, we found that GSK3-β is phosphorylated; furthermore, GATA4 shows a nuclear localization on immunohistochemical staining, which is thus in line with results from isolated cardiomyocytes. Our results also strongly suggest that Akt is a mediator of the β-AR effects on myocardial contractility. Usually, a marked increase in cardiomyocyte cell size by a transgenic approach is accompanied by a decrease in cardiac function. In contrast, in this transgenic mouse model, the basal LV dP/dt_max, a reliable index of inotropy in the absence of acute changes in preload (19, 20), was increased by 67% in transgenic mice. The lack of enhancement of indices of LV relaxation at rest in the Akt transgenic mice (which usually accompanies increases in contractility) can likely be attributed to the substantial cardiac hypertrophy, because it has been shown that LV relaxation can be impaired in association with cardiac hypertrophy when myocardial contractility is normal (21). It seems unlikely that there was impaired LV filling under basal conditions in the transgenic mice in view of the lack of increased fibrosis and normal LV end-diastolic pressure. However, LV relaxation, assessed by LV dP/dt_min, was impaired in these mice during dobutamine infusion at high drug dosage. Our data thus support the view that the lack of a change in basal relaxation in vivo is caused by the combination of enhanced contractility and the effect of hypertrophy in the intact LV. This effect did not alter the survival of the E40K mice as compared with control nontransgenic littermates at 1 1/2 yr of life.

Overexpression of Akt does not affect the efficiency of β-AR binding to its ligand. In fact, β-AR density, affinity, and capacity to generate cAMP were not affected by Akt overexpression in transgenic mice as compared with control mice. Thus, although Akt is a target of β-AR, its activation is not involved in the regulation of β-AR activity.

In this study, we cannot rule out that Akt modulates protein levels of the major intracellular calcium regulatory proteins (SERCA2, phospholamban, Ca²⁺/Na⁺ exchanger, ryanodine receptor). Whether Akt induces posttranscriptional modification of these proteins cannot be excluded either. Our work strongly suggests that Akt acts on calcium metabolism, although the level of regulation still remains uncertain. In fact, our results do not discriminate between an effect on the calcium channel or on SERCA activity. The shift upward in the dose-response of LV dP/dt_max to β-AR stimulation, with a response to the maximum dose that is comparable to the peak dobutamine response in controls, resembles that observed in phospholamban knockout mice (22), and it is possible that Akt regulates calcium release and uptake from the sarcoplasmic reticulum. On the other hand, it is also possible that Akt acts on the calcium channel or on the ryanodine receptor, leading to an increase in SERCA2 activity. Further work will clarify this issue. In any case, the remarkable actions of Akt in vivo to induce enhanced myocardial contractility and cell size may carry implications for treatment of heart failure by a mechanism downstream from the β-AR.

Acknowledgments

We thank Dr. Philip Tsichlis for his thoughtful comments. This research was supported by grants from the American Heart Association, Associazione Italiana per la Ricerca sul Cancro, Fondi 1% Ministero della Sanita' (to G.C.), and the National Institutes of Health (to C.M.C.).

Abbreviations

PI3K, phosphatidylinositol 3-kinase
β-AR, β-adrenergic receptor
GSK3, glycogen synthase kinase3
ISO, isoproterenol
MAPK, mitogen-activated protein kinase
LV, left ventricular

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