Mechanism of Enhanced Cardiac Function in Mice with Hypertrophy Induced by Overexpressed Akt*

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3–4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 ± 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 ± 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 ± 0.2 versus 3.5 ± 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 ± 50 versus 250 ± 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 ± 15 versus 98 ± 18 μm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 ± 0.3 versus 7.2 ± 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.

Besides these well characterized functions of Akt, transgenic mice with cardiac-specific overexpression of constitutively active Akt have increased base-line contractility, namely an elevated LV 1 dP/dt max (11). However, the cellular mechanism responsible for increased myocardial function by Akt activation remains unknown. In this study, we examined the correlation of in vivo cardiac function with in vitro intrinsic myocyte contraction and relaxation. To determine the cellular mechanism of the enhanced cardiac function in TG, we examined L-type Ca 2ϩ channel function and Ca 2ϩ -handling proteins, including sarcoplasmic reticulum (SR) Ca 2ϩ ATPase 2a (SERCA2a), phospholamban (PLB), calsequestrin, the ␣-subunit of the Ltype Ca 2ϩ channel (␣ 1c ), and Na ϩ /Ca 2ϩ exchanger (NCX), as well as myocardial ryanodine receptor binding. Our results suggest that activation of Akt enhances Ca 2ϩ transients and facilitates both contraction and relaxation in isolated cardiac myocytes, which is associated with enhanced Ca 2ϩ influx and increased protein levels of SERCA2a. These features indicate a novel function of Akt in the mouse heart.

EXPERIMENTAL PROCEDURES
Transgenic Mice-The generation of transgenic mice with cardiacspecific overexpression of constitutively active Akt (E40K Akt) (TG) has been described (11). We have also generated transgenic mice with * This study was supported in part by National Institutes of Health Grants HL33065, HL33107, HL59139, HL61476, HL62442, HL65182, HL65183, HL67724, HL69020, and AG14121; American Heart Association Grants 0030125N and 9950673N; and Fondi Ministero Salute, Fondi Italia-USA, and Associazione Italiana per la Ricerca sul Cancro. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  cardiac-specific overexpression of kinase-inactive Akt (Tg-KI-Akt). Tg-KI-Akt did not exhibit an obviously abnormal cardiac phenotype, and cardiac function was apparently normal. In this study, 3-5-month-old TG, Tg-KI-Akt, and nontransgenic littermate mice (WT) were used. Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (37).
Myocytes were field-stimulated at 1 Hz, and contraction was measured using a video motion edge detector (VED103, Crescent Electronics) as described previously (19). The external solution contained 1 mM Ca 2ϩ . The following contractile properties were calculated from length data: % contraction and the rate of shortening (ϪdL/dt). Relaxation properties (ϩdL/dt, the rate of relengthening; t R 70%, the time for 70% relaxation) were also assessed. SR Ca 2ϩ reuptake function was examined in response to thapsigargin (10 Ϫ10 , 10 Ϫ9 , and 10 Ϫ8 M) (Sigma) in both WT and TG. Cells were loaded with 5 M Fura-2 AM (Sigma), and Ca 2ϩ transients were measured using the Photoscan dual beam spectrofluorophotometer (Photon Technology) (19).
Western Blot Analysis for SERCA2a, PLB, Calsequestrin, ␣ 1c , and NCX-Left ventricular tissue was obtained and immediately frozen in liquid nitrogen (Ϫ80°C). On the day of the experiment, tissue samples were homogenized in 0.75 M NaCl, 10 mM histidine (pH 7.5) with protease, phosphatase, and kinase inhibitors. Equal amounts of protein were dissolved in 1% SDS, 100 mmol/liter Tris-HCl (pH 6.5), 10% glycerol, 0.05% bromphenol blue, 5% 2-mercaptoethanol, separated by 12.5% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. Blots for SERCA2a and PLB were incubated for 30 min at room temperature with a 1:20,000 dilution of rabbit anti-SERCA2a polyclonal antibody (generous gift from Dr. Frank Wuytack, Leuven, Belgium) or 1:20,000 mouse anti-PLB monoclonal Ab (Affinity Bio-Reagents Inc., Golden, CO) in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Blots for calsequestrin were incubated overnight at 4°C with a 1:25,000 dilution of rabbit anti-canine cardiac calsequestrin polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) in the same buffer. Blots for NCX were incubated overnight at 4°C with a 1:1,000 dilution of monoclonal anti-NCX (Swant) in Tris-buffered saline and 1% milk. Blots for ␣ 1c were incubated overnight at 4°C with a 1:500 dilution of anti-␣ 1c antibody (Affinity BioReagents) in Trisbuffered saline and 1% milk. Intensities of the bands were evaluated by densitometric scanning using a Personal Densitometer SI with Image-QuaNT software (Amersham Biosciences) and normalized for protein loading.
Alkaline Phosphatase Treatment-Cell lysates (15 g) were treated with a one-ninth volume of 10ϫ alkaline phosphatase buffer (Promega, Madison, WI) at 30°C for 10 min and then treated with 30 units of calf intestine alkaline phosphatase at 30°C for 10 min. 4ϫ SDS-PAGE loading buffer was added, and the samples were separated by 8% SDS-PAGE. Then, SERCA2a protein was processed for Western blotting as described above.
Ryanodine Receptor Binding Studies-Ryanodine receptor binding studies were conducted as described previously (20). Assays were performed using 6 concentrations of [ 3 H]ryanodine (1 to 30 nmol/liter) and 100 g of membrane protein in HEPES buffer (with 0.3 mmol/liter CaCl 2 ) in a total volume of 150 l. Unlabeled ryanodine (10 mol/liter) was used to determine nonspecific binding. Incubation was at 37°C for 90 min. All assays were performed in triplicate and terminated by rapid filtration through Whatman GF/F filters washed with 4 ml of cold buffer (150 mmol/liter KCl, 10 mmol/liter Tris HCl, pH 7.4). Filters were vortexed in 5 ml of Ecoscint and counted in a beta scintillation counter for 5 min. All assays were standardized by the protein content. Protein concentrations were determined using the BCA method. A linear regression was performed on the amount bound versus bound/free ligand.
Gene Expression by Reverse Transcription-PCR-Under anesthesia, the mouse heart was removed, frozen in liquid nitrogen, and stored at Ϫ80°C. The tissue was homogenized in a solution containing guanidine isothiocyanate in the presence of 12 g of Escherichia coli rRNA as a carrier, and total RNA was isolated by the CsCl gradient technique. After ultracentrifugation at room temperature in a TL100 (Beckman) at 48,000 ϫ g for 19 h, the supernatant was removed and the RNA pellet was dissolved in water. Sodium acetate (pH 5.3) was added to the solution to a final concentration of 3 M, and the RNA was precipitated with absolute ethanol. The RNA was then rinsed in 70% ethanol, vacuum-dried, and re-dissolved in water. cDNA was synthesized from 2 g of total RNA by using random primers and reverse transcriptase according to the manufacturer's instructions (Roche Applied Science).  (17). The PCR products were fractionated on 2% agarose and visualized by ethidium bromide staining.
Preparation of Adult Cardiac Myocyte Culture and Adenovirus Transfection-Adult rat cardiac myocyte cultures were prepared as described previously (22) to determine whether overexpression of Akt enhances the expression of SERCA protein as a mechanism responsible for the enhanced myocyte contractile and relaxation function. Adenovirus harboring either Akt (multiplicity of infection 10) or LacZ (multiplicity of infection 10) was applied 2 h after myocyte isolation. Methods for adenovirus transfection have been described (7).
Statistical Analysis-All data are expressed as mean Ϯ S.E. A comparison of the data among the groups was made by analysis of variance or by Student's t test, and statistical significance was taken at p Ͻ 0.05. The myocyte data were averaged for each animal for statistical comparison.

RESULTS
Echocardiography-Heart weight/body weight ratio was increased (p Ͻ 0.01) in TG (11.5 Ϯ 1.4 mg/g) compared with WT (7.2 Ϯ 0.4 mg/g). The results of echocardiographic measurements are summarized in Table I. Heart rate was similar between TG and WT. The end diastolic wall thickness was increased (p Ͻ 0.01) in TG (0.84 Ϯ 0.06 mm) compared with WT (0.62 Ϯ 0.03 mm). Although end diastolic dimensions were similar, end systolic dimensions were significantly reduced by 15% in TG compared with WT (p Ͻ 0.05). The LV ejection fraction was significantly enhanced (p Ͻ 0.01) in TG (84 Ϯ 2%) compared with WT mice (73 Ϯ 1%). These results suggest that TG have concentric hypertrophy with increased systolic function.
Myocyte Contraction and Ca 2ϩ Transients- Fig. 1 shows representative contraction/relaxation and Ca 2ϩ transient record- To examine whether the enhanced contraction and relaxation in TG myocytes are caused by changes in intracellular Ca 2ϩ levels, we measured Ca 2ϩ transients. As shown in Fig. 1 and Table II, the amplitude of Ca 2ϩ transients in TG myocytes was significantly increased (396 Ϯ 50 versus 250 Ϯ 24 nmol/ liter, p Ͻ 0.05). There were no significant differences in levels of diastolic free Ca 2ϩ concentration (181 Ϯ 11 versus 171 Ϯ 14 nmol/liter, p ϭ not significant). The time for 70% Ca 2ϩ reuptake was also accelerated in TG myocytes compared with WT myocytes (78 Ϯ 6 versus 141 Ϯ 8 ms, p Ͻ 0.05). Thus, both peak Ca 2ϩ levels and Ca 2ϩ uptake are increased in myocytes isolated from TG. Myocytes isolated from Tg-KI-Akt did not show any significant changes in peak Ca 2ϩ levels and Ca 2ϩ uptake.
These results indicate that changes in contractile function and Ca 2ϩ transients are dependent upon Akt activity.
L-type Ca 2ϩ Channel Currents-Because Ca 2ϩ influx through L-type Ca 2ϩ channels plays an essential role in excitation-contraction coupling, we examined L-type Ca 2ϩ currents (I Ca ) in LV myocytes isolated from TG and WT (Fig. 2). LV myocyte size estimated by cell capacitance was significantly larger in TG myocytes compared with WT myocytes (Fig. 2B), consistent with the heart weight/body weight ratio data.
The traces in Fig. 2 show typical I Ca recorded from TG and WT myocytes. In both groups, I Ca was activated around Ϫ30 mV and reached its maximum near ϩ10 mV. However, the peak I Ca amplitude, normalized by the cell capacitance (pA/pF) was significantly larger (p Ͻ 0.01) in TG myocytes (9.0 Ϯ 0.3 pA/pF) compared with WT myocytes (7.2 Ϯ 0.3 pA/pF). The inactivation kinetics of I Ca at ϩ10 mV was significantly faster in TG myocytes compared with WT myocytes (Fig. 2B). In mouse ventricular myocytes, I Ca inactivation is controlled by Ca 2ϩ in the sarcolemmal subspace (19,23,24). To examine whether the difference in I Ca inactivation rate between WT and TG myocytes was due to the contribution of Ca 2ϩ released from the SR, the SR Ca 2ϩ content was depleted by ryanodine (10 M). Following the application of ryanodine, the rate of I Ca inactivation as measured by t1 ⁄2 was significantly increased in both WT and TG myocytes, and no significant difference in the inactivation rate was detected between two groups (31.4 Ϯ 2.3 ms, n ϭ 6 versus 29.5 Ϯ 2.1 ms, n ϭ 6).
The results indicate that the faster I Ca inactivation observed in TG myocytes might be related to enhanced SR Ca 2ϩ release (See below).
SERCA2a, PLB, Calsequestrin, ␣ 1c , NCX, and Ryanodine Receptor Protein-Because both enhanced amplitude and relaxation of the Ca 2ϩ transient may be caused by changes in SR Ca 2ϩ -handling proteins, the expression of these proteins was determined by immunoblot analyses. The protein expression of Akt in myocardium was increased Ϸ25-fold in TG compared with WT mice. SERCA2a protein levels were increased significantly (p Ͻ 0.05) by 6.6-fold in TG compared with WT mice. In contrast, protein levels of PLB, calsequestrin, ␣ 1c , and NCX were not significantly different between TG and WT mice (Fig.  3, A and B). The affinity and number of ryanodine receptors were similar between WT and TG (K D ϭ 7.65 versus 6.95 nM; B max ϭ 499 versus 511 fmol/mg in WT and TG mice).
Semi-quantitative reverse transcription-PCR measurements indicated that there was no significant difference in SERCA2a mRNA levels between TG and WT (Fig. 3C). Thus, the increase in SERCA2a protein in TG occurs post-transcriptionally.

Overexpression of Akt Increases Expression of SERCA2a in Adult Ventricular Cardiac Myocytes-To determine whether transient overexpression of Akt is sufficient to increase
SERCA2a protein expression in cardiac myocytes, we conducted adenovirus-mediated overexpression of Akt in primary cultured adult rat ventricular cardiac myocytes. Forty-eight hours after transfection, overexpression of Akt was confirmed by immunoblot analyses. Immunoblotting of the same filters with anti-SERCA2a antibody indicated that SECA2a expression was enhanced by 4.8-fold (n ϭ 4, p Ͻ 0.05) compared with control virus-transfected cardiac myocytes (Fig. 4). Interestingly, although SERCA2a was detected in two bands in control virus-transfected myocytes, only a slower migrating form, possibly a phosphorylated form of SERCA2a, was detected in Akt virus-transfected myocytes. In fact, the slower migrating band disappeared after phosphatase treatment, consistent with the notion that it represents a phosphorylated form of SERCA2a (data not shown).

Up-regulation of SERCA2a Increases Contractile and Relaxation Function in Isolated Cardiac
Myocytes from TG-We examined the effect of thapsigargin, a specific inhibitor of SERCA2a, on contraction of isolated myocytes. The dose of thapsigargin inhibiting % contraction by half (IC 50 ) was 2.6fold higher in myocytes isolated from TG than in those from WT (Fig. 5). Enhanced relaxation function in TG myocytes was also abolished after thapsigargin (10 Ϫ10 M). These results are con-sistent with the notion that enhanced SERCA2a plays an important role in mediating increased cardiac myocyte contraction and relaxation in TG. DISCUSSION Akt plays a central role in glucose metabolism, cell growth, angiogenesis, transcription, apoptosis, and protein synthesis (25). We found that overexpression of constitutively active Akt  2. A, whole-cell I Ca recorded in WT and TG myocytes. Traces show currents elicited from a holding potential of Ϫ50 mV to the indicated test potentials. TG myocytes had larger I Ca amplitudes and faster inactivation kinetics. B, average cell capacitance, I Ca density, and half-maximal decay (t1 ⁄2 ) of I Ca obtained from WT and TG myocytes. Data points are mean Ϯ S.E. The numbers correspond to total number of cells measured. An asterisk indicates that the mean values are significantly different (p Ͻ 0.01) from respective WT controls. in the mouse heart increased both LV ejection fraction, in vivo, and isolated myocyte contraction in vitro. A recent study using the same TG model demonstrated increased LV dP/dt with a nonsignificant increase in LV ejection fraction (11). The failure to observe significantly increased LV ejection fraction in the prior study (11) may be due to heart rate, which was significantly lower in TG than WT, whereas the heart rates were similar in the two groups in the present study. Interestingly, the improved LV function occurred in the presence of significant LV hypertrophy, which is thought to reduce LV function. The goals of the present study were: 1) to investigate whether constitutively active Akt has direct effects upon contractility and Ca 2ϩ handling in isolated cardiac myocytes; and if so, 2) to identify the downstream mechanism of the enhanced cardiac myocyte function by Akt activation.
We have demonstrated that the enhanced cardiac function in TG in vivo is associated with increased intrinsic contractile and relaxation function in isolated cardiomyocytes in vitro. Moreover, the increased contraction and relaxation in myocytes isolated from TG paralleled the increased peak systolic amplitude and more rapid diastolic decay in intracellular Ca 2ϩ transients. The studies in isolated myocytes also demonstrated increased Ca 2ϩ channel currents. These functional changes in isolated myocytes were not observed in Tg-KI-Akt, suggesting that they are dependent upon the kinase activity of Akt. This indicates that activation of Akt directly affects contraction and relaxation through changes in Ca 2ϩ handling in individual cardiac myocytes.
It should be noted that the enhanced cardiac contractility was not observed in TG mice with cardiac-specific overexpression of constitutively active PI3K␣, an upstream regulator of Akt (26), or in other forms of constitutively active Akt (T308D, S473D-Akt, and myr-Akt) (10,27) distinct from the one used in this study (E40K-Akt), despite the fact that all mice exhibited cardiac hypertrophy. Furthermore, phosphatidylinositol 3-kinase-␥ activated by inhibition of PTEN (phosphatase and tensin homologue deleted on chromosome 10) negatively regulates cardiac contractility (28). Although myr-Akt is constitutively localized at the plasma membrane, both wild type Akt and E40K-Akt undergo growth factor-dependent membrane translocation (29). These results suggest that cardiac contractility may be tightly regulated by activation of a specific component of the phosphatidylinositol 3-kinase pathway and/or by subcellular localization or the substrate specificity of the Akt mutants used. Thus, it is possible that E40K and wild type Akt may share downstream targets for the enhanced cardiac function.
To identify the downstream mechanisms of Akt responsible for enhanced myocyte contraction and relaxation, we examined mechanisms known to control intracellular Ca 2ϩ transients. We found that the amplitude of I Ca was enhanced in Akt myocytes by Ϸ20% compared with that in WT myocytes, which may be at least in part responsible for the enhanced cellular Ca 2ϩ transients. Akt mediates insulin-like growth factor-induced potentiation of I Ca in neuronal cells (30). Therefore, to determine whether the increased I Ca was due to altered Ca 2ϩ channel expression, we measured the expression level of the ␣-subunit of the cardiac L-type Ca 2ϩ channel (␣ 1c ) and found no significant differences in ␣ 1c protein expression between WT and TG. These results indicate that the enhanced I Ca amplitude observed in Akt myocytes may be related to the altered channel regulation, i.e. an increase in open probability or con- ductance of L-type Ca 2ϩ channels through increased phosphorylation, rather than protein expression.
Ca 2ϩ entry through L-type Ca 2ϩ channels is a critical first step in the Ca 2ϩ -handling cascade. The cytosolic Ca 2ϩ transients and contraction elicited by membrane depolarization are strongly influenced by the amount of Ca 2ϩ influx through the channel. Thus, an increase in Ca 2ϩ entry through the L-type Ca 2ϩ channels is likely to contribute to the increase in cellular Ca 2ϩ transients observed in TG myocytes (31). It is also possible that Akt could alter the Ca 2ϩ dependence of the open probability of the ryanodine receptor. Our results, however, suggest that neither the affinity nor the total number of ryanodine receptors was affected by activation of Akt.
In normal cardiac myocytes isolated from rats or mice, Ϸ90% of cytosolic Ca 2ϩ is removed through SERCA (32). Overexpression of SERCA2a or the enhanced SERCA activity by phosphorylation of PLB accelerates Ca 2ϩ transients and cardiac relaxation (33,34). In the present study, SERCA2a protein levels were up-regulated in TG mice. Interestingly, we found that higher doses of thapsigargin are required to reduce % contraction in cardiac myocytes isolated from TG and the enhanced relaxation function in TG myocytes was abolished in the presence of thapsigargin. These results strongly suggest that increased levels of SERCA2a lead to enhanced Ca 2ϩ transients and contraction as well as accelerated relaxation in cardiac myocytes. Our results indicate that up-regulation of SERCA2a expression by Akt is mediated by post-transcriptional mechanisms. The amino acid sequence of mouse SERCA2a contains potential phosphorylation sites by Akt. Our experiments using cultured adult ventricular cardiac myocytes indicated that adenovirus-mediated transient overexpression of Akt increased expression of SERCA2a. Furthermore, on the SDS-PAGE, only a slower migrating form of SERCA2a appeared after Akt overexpression. This slower migrating form disappeared after phosphatase treatment, and thereafter only the faster migrating form was observed (data not shown), consistent with the concept that SERCA2a is phosphorylated, when Akt is overexpressed. Whether SERCA2a is a physiological substrate of Akt, and if so, whether activation of Akt enhances translation or stability of SERCA2a, remains to be elucidated.
We and others have shown that activation of Akt causes cardiac myocyte hypertrophy both in vitro and in vivo (7,10,11,27). Because ventricular hypertrophy is usually accompanied by decreases in SERCA2a, which lead to impairment of Ca 2ϩ handling, up-regulation of SERCA2a and acceleration of Ca 2ϩ transients despite the induction of hypertrophy in this transgenic model is unique, particularly in view of the well known effects of hypertrophy in interfering with Ca 2ϩ handling and myocyte contraction (35,36). Because Akt prevents cardiac myocyte death in response to pathologic stimuli (16), stimulation of Akt might be an ideal method of enhancing the contractility of the heart and improving Ca 2ϩ handling in response to pressure overload.
In summary, transgenic overexpression of protein kinase Akt enhances myocyte contractility and relaxation through acceleration of intracellular Ca 2ϩ transients. The underlying cellular mechanism(s) include potentiation of L-type Ca 2ϩ channel function and up-regulation of SERCA2a.