Published online before print November 17, 2005, doi: 10.1161/01.RES.0000196568.11624.ae
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© 2005 American Heart Association, Inc.
Integrative Physiology |
Nuclear Targeting of Akt Enhances Ventricular Function and Myocyte Contractility
From the Cardiovascular Research Institute (M.R., A.B., K.U., M.E.P.-I., T.J.K., G.F., E.H.S., E.M., A.L., P.A.), Department of Medicine, New York Medical College, Valhalla; Cardiovascular Research Center (H.K., S.R.H.), Temple University, Philadelphia, Pa; and San Diego State University Heart Institute (M.A.S.), Calif; and Tardini-Vitali-Mazza-Olivetti Stem Cell Center (E.M.), Parma, Italy.
Correspondence to Piero Anversa, MD, Cardiovascular Research Institute, Vosburgh Pavilion, Rm 302, New York Medical College, Valhalla, NY 10595. E-mail piero_anversa{at}nymc.edu
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Abstract |
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Cytoplasmic overexpression of Akt in the heart results in a myopathy characterized by organ and myocyte hypertrophy. Conversely, nuclear-targeted Akt does not lead to cardiac hypertrophy, but the cellular basis of this distinct heart phenotype remains to be determined. Similarly, whether nuclear-targeted Akt affects ventricular performance and mechanics, calcium metabolism, and electrical properties of myocytes is unknown. Moreover, whether the expression and state of phosphorylation of regulatory proteins implicated in calcium cycling and myocyte contractility are altered in nuclear-targeted Akt has not been established. We report that nuclear overexpression of Akt does not modify cardiac size and shape but results in an increased number of cardiomyocytes, which are smaller in volume. Additionally, the heart possesses enhanced systolic and diastolic function, which is paralleled by increased myocyte performance. Myocyte shortening and velocity of shortening and relengthening are increased in transgenic mice and are coupled with a more efficient reuptake of calcium by the sarcoplasmic reticulum (SR). This process increases calcium loading of the SR during relengthening. The enhanced SR function appears to be mediated by an increase in SR Ca2+-ATPase2a activity sustained by a higher degree of phosphorylation of phospholamban. This posttranslational modification was associated with an increase in phospho–protein kinase A and a decrease in protein phosphatase-1. Together, these observations provide a plausible biochemical mechanism for the potentiation of myocyte and ventricular function in Akt transgenic mice. Therefore, nuclear-targeted Akt in myocytes may have important implications for the diseased heart.
Key Words: Akt • myocyte mechanics • myocyte size and number
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Introduction |
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Protein kinase B, also referred to as Akt, phosphorylates multiple cytoplasmic and nuclear substrates implicated in cell survival and growth of several organs including the heart.1 Although myocyte survival and cellular hypertrophy may be viewed as important adaptations of the overloaded heart against the onset of ventricular decompensation,2 the targeted expression of constitutively activated Akt to the myocardium has resulted in cardiac hypertrophy3–7 and ventricular dysfunction.6 In these cases, however, transgene activity was widespread throughout cardiomyocytes at nonphysiological levels, raising the possibility that the nuclear accumulation of Akt may retain the antiapoptotic effects of this serine–threonine kinase, without promoting organ hypertrophy and alterations in cardiac performance. In this regard, hearts of mice expressing nuclear-targeted Akt show no evidence of myopathy8 in contrast to other cardiac-specific Akt transgenics created with constitutively activated kinase. Targeting of Akt to myocyte nuclei preserves cell viability through the phosphorylation of survival factors within the nucleus that interfere with apoptotic death signaling.1,8–11 Thus, whether Akt is expressed in the cytoplasm or in the nucleus, cardiomyocyte apoptosis is inhibited, but whether the absence of cardiac hypertrophy in the latter condition affects differently ventricular hemodynamics is a relevant unanswered question.
In the models of Akt-induced cardiac hypertrophy, the analysis of myocardial and/or myocyte contractility cannot discriminate the effects of increased cell size from those related to the overexpression of Akt on the mechanical properties of the heart and its parenchymal cells.7,12 Similarly difficult is the interpretation of changes in sarcoplasmic reticulum (SR) Ca2+ ATPase2a (SERCA2a) and other biochemical parameters in hypertrophied myocytes from Akt transgenic mice.12 Additionally, the risk of provoking cardiac hypertrophy, a major factor of heart failure in humans,13 precludes the feasibility of genetic manipulation of the heart with cytoplasmically overactive Akt constructs. Therefore, we have defined the cardiac phenotype of a transgenic mouse in which Akt was localized to cardiomyocyte nuclei with the Akt/nuc transgene driven by the mouse 
-MHC promoter.8 Measurements of the anatomical and functional properties of the hearts were complemented with the analysis of the mechanical and electrical behavior of cardiomyocytes. Finally, the biochemical characteristics of Ca2+ handling regulatory proteins and myofilament protein subunits were determined to obtain information about the physiological implications of Akt in myocardial performance.
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Materials and Methods |
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Transgenic (TG) and wild-type (WT) mice were studied anatomically, functionally, and biochemically at 3 months of age. Electrophysiological and mechanical parameters were obtained in isolated myocyte preparations.
An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
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Results |
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Cardiac Anatomy and Myocyte Size and Number
The weight of the diastolic arrested heart and the weights of the left and right ventricle did not differ in TG and WT. Body weight was comparable in the 2 groups resulting in a similar heart weight-to-body weight ratio (Figure 1A). Left ventricular (LV) free wall thickness, chamber diameter, longitudinal axis, and volume were not different between TG and WT. Therefore, the wall thickness-to-chamber radius ratio and the LV mass-to-chamber volume ratio did not vary in the 2 groups of mice.
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The volume of mononucleated, binucleated, trinucleated, and tetranucleated LV myocytes was determined by optical sectioning of isolated cells by confocal microscopy.14 By this approach (Figure 1B), the distribution of each myocyte class in TG and WT was obtained (Figure 1C). Although there was some overlap in the range of volumes of mononucleated and binucleated myocytes in TG and WT, cells in TG were shifted to the left to smaller sizes. In TG, the average volume of mononucleated and binucleated myocytes was 27% and 20% smaller than in WT, respectively (Figure 1D). However, the volume of trinucleated and tetranucleated myocytes was not different between TG and WT. Importantly, binucleated myocytes constituted &92%, mononucleated &6%, and multinucleated &2% of all cells in both animal groups. The number of mononucleated and binucleated myocytes in the LV of TG mice was 60% and 26% higher than in WT, respectively. The number of multinucleated myocytes was similar in TG and WT; together, there were 27% more LV myocytes in TG than in WT (Figure 1D). Additionally, TG myocytes had a 10-fold increase in total Akt protein but phospho-Akt at Ser473 increased only in nuclei. This was consistent with the comparable level of expression of PI3K (phosphatidylinositol 3-kinase) in TG and WT myocytes (Figure 1E and 1F). Therefore, nuclear-targeted Akt does not alter cardiac size and shape but results in an increased number of myocytes, which are smaller in volume.
Ventricular Function
Echocardiographic parameters were obtained in unanesthetized, unrestrained mice, whereas hemodynamic data were collected under anesthesia in closed-chest preparation. Echocardiographically, LV diastolic volume was similar in TG and WT, but LV systolic volume was smaller in TG than in WT, resulting in a significant increase in ejection fraction in TG (Figure 2). Hemodynamically, LV end-diastolic pressure was comparable in the 2 groups of mice. However, LV systolic pressure, LV developed pressure, and LV +dP/dt and –dP/dt were higher in TG than in WT (Figure 2). The pressure values in combination with the anatomical measurements of wall thickness and chamber radius allowed us to compute midwall systolic and diastolic stress. Diastolic and systolic stress did not differ between TG and WT. Therefore, nuclear-targeted Akt overexpression is characterized by enhanced cardiac function.
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Myocyte Mechanics and Ca2+ Transients
Isolated myocytes were field stimulated at 1-Hz pacing rate and their mechanical properties were determined by using a video-edge track detection system.15 LV myocytes from TG mice showed enhanced contractile function (Figure 3A and 3B). In comparison with WT myocytes, TG myocytes had an 18% increase in fractional shortening, a 20% increase in maximal rate of contraction (–dL/dt), and a 28% increase in maximal rate of relaxation (+dL/dt). Timing parameters of contraction were similar in both groups of cells, but velocity of shortening and relengthening was faster in TG myocytes.
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P<0.05 vs Tyrode.To define the mechanisms underlying the potentiated myocyte contractility associated with nuclear-targeted Akt, intracellular Ca2+ handling was analyzed.16 Myocytes from TG and WT were loaded with Fluo-3 and were field stimulated at 1 Hz (Figure 3C). Ca2+ transient amplitude was comparable in TG and WT myocytes, but Ca2+ decay was faster in transgenics. TG myocytes showed a 15% and 12% decrease in the time required to reach 50% and 90% baseline fluorescence, respectively. Moreover, the time constant of Ca2+ decay measured by monoexponential fitting of the data were reduced by 29% in TG myocytes (Figure 3D). Myocytes were then treated with caffeine to measure Ca2+ transients under the condition of maximum release of this cation from the SR. Caffeine abolishes the regulatory role of ryanodine receptor (RyR) channels in the release of Ca2+ from the SR. In the presence of caffeine, the amplitude of Ca2+ transients was 25% higher in TG than in WT (Figure 3E), indicating that Ca2+ loading of the SR was increased in TG myocytes. Therefore, Akt overexpression in myocyte nuclei does not enhance the amplitude of Ca2+ transients but appears to be coupled with a more efficient reuptake of Ca2+ by the SR during relengthening.
Stimulation Frequencies, Ca2+ Transients, and Sarcomere Mechanics
To determine whether TG myocytes possessed a better functioning SR than WT myocytes, Ca2+ handling and sarcomere shortening were measured simultaneously. Different rates of stimulation were used to assess the rate dependency of intracellular developed Ca2+ and myocyte contractility. TG and WT myocytes responded differently to an increase in pacing rate from 0.5 Hz to 1 Hz and 2 Hz (Figure 4A). Ca2+ transient amplitude and sarcomere shortening at 0.5 Hz were measured, and the corresponding values at 1 and 2 Hz were normalized with respect to those detected at 0.5 Hz. This was done to have a uniform reference point for comparison and to express the data at higher frequency as a percent change of the results at 0.5 Hz. A negative percent change in Ca2+ transients was detected in WT myocytes when they were stimulated at 1 and 2 Hz. Conversely, a positive percent change in Ca2+ transients was seen in TG myocytes at 1 and 2 Hz (Figure 4B). Similarly, there was a consistent decrease in sarcomere shortening in WT myocytes and a consistent increase in sarcomere shortening in TG myocytes with increasing frequency of stimulation.
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The distinct changes in Ca2+ transients of WT and TG myocytes with rates of stimulation can reflect different changes in the amount of Ca2+ available for release from the SR, alterations in the trigger of Ca2+ release, modifications in the process of Ca2+ release, or a combination of these phenomena.17 These possibilities correspond, respectively, to Ca2+ loading of the SR, kinetics of L-type Ca2+ channels, and opening of RyR channels. For this purpose, the stimulation of myocytes at 2 Hz was followed by a test pulse that was applied after a 2-sec pause. This pause period relieves a potential inactivation of the L-type Ca2+ channels and/or RyR channels.18 In both WT and TG myocytes, Ca2+ transient amplitude and peak were comparable before and after the pause (Figure 4C), indicating that the release of Ca2+ at this pacing rate was not affected by the kinetics of L-type Ca2+ and RyR channels. Therefore, the differential response of WT and TG myocytes to the increased rates of stimulation is most likely dependent on the enhanced Ca2+ loading of the SR in TG myocytes.
L-type Ca2+ Current and RyR Channels
Ca2+ influx from the extracellular compartment to the inside of the cell via L-type channels plays a crucial role in excitation–contraction coupling by triggering the release of Ca2+ from the SR through the activation of the RyR channels.19 Although Ca2+ loading of the SR was greater in TG myocytes than in WT myocytes, Ca2+ transients were similar in the 2 groups of cells. The high level of Ca2+ in the SR of TG myocytes, in the absence of a corresponding increase in Ca2+ transients, raised the possibility that a defect may be present in the L-type Ca2+ current (ICaL), which triggers the release of Ca2+ from its site of storage. Alternatively, the density and phosphorylation state of the RyR channels may be lower in TG myocytes attenuating Ca2+ flux from the SR to the cytoplasm.17 Therefore, patch-clamp experiments were performed20 to characterize ICaL.
The density of ICaL was tested at different potentials in WT and TG myocytes to obtain current–voltage relation curves; these current–voltage curves were essentially identical in the 2 groups of myocytes (Figure 5A). Similarly, the voltage dependency of the activation and inactivation of L-type Ca2+ current was comparable in TG and WT myocytes (Figure 5B). Importantly, the activation and inactivation curves were fitted with Boltzmann functions to establish quantitatively their apparent superimposition. By this approach, we determined that the half-maximal activation and inactivation potentials of the current in TG and WT myocytes were not statistically different (Figure 5B).
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The inactivation time course of the L-type Ca2+ current was fitted by a biexponential function with comparable time constants for the 2 groups of myocytes (Figure 5C). Recovery from inactivation of ICaL was evaluated by the use of a double-pulse protocol with a variable interpulse duration. With this procedure, it was possible to establish the time necessary for the complete recovery of ICaL (Figure 5D). The plotted curves reflecting the fractional recovery of ICaL versus the interpulse duration were found to be similar in TG and WT myocytes.
To determine whether the release of Ca2+ from the SR was conditioned by the properties of the RyR channels, the density of these channels was measured by confocal microscopy and fluorescence intensity (Figure 5E). The level of expression and phosphorylation of RyR was not different between TG and WT myocytes (Figure 5F and supplemental Figure I). Therefore, the mechanisms that trigger the release of Ca2+ from the SR and modulate Ca2+ flux from the SR to the cytoplasm are similar in TG and WT myocytes.
SERCA2a, Phospholamban, and Na+–Ca2+ Exchange
SERCA2a and the Na+–Ca2+ exchange (NCX) are the predominant contributors of the influx of Ca2+ into the SR and the extrusion of Ca2+ from the SR to the extracellular compartment, respectively. The mitochondrial Ca2+ uniporter is involved in a small reuptake of Ca2+ from the cytoplasm and the sarcolemmal Ca2+-ATPase participates modestly in the transport of Ca2+ outside of the cell.17 Phospholamban (PLB) regulates the function of SERCA2a, and the nonphosphorylated form of PLB inhibits SERCA2a and thereby the reuptake of Ca2+ by the SR. Conversely, the phosphorylated form of PLB promotes the role of SERCA2a and the transport of Ca2+ into the SR.21 Because of the major function that SERCA2a-PLB plays in the reuptake of Ca2+ into the SR (&92%) and NCX in the extrusion of Ca2+ to the extracellular space (&7%) during relengthening, these systems were characterized biochemically. This was done in an attempt to identify the molecular basis of the enhanced Ca2+ transient decay and increased Ca2+ loading of the SR in myocytes with nuclear-targeted Akt.
The quantity of SERCA2a was not different between TG and WT myocytes by Western blotting (Figure 6A) or immunocytochemistry (Figure 6B). Similarly, the amount of PLB was comparable in TG and WT myocytes (Figure 6C and 6D), but the level of phosphorylation of the mono- and pentameric forms of PLB at Ser16 was increased in TG myocytes (Figure 6E and 6F). NCX protein increased in TG myocytes (Figure 6G and supplemental Figure II), but this effect on Ca2+ metabolism was blunted by enhanced PLB function. Together, these observations indicate that the faster decay of Ca2+ in TG myocytes is mediated by PLB phosphorylation and increased Ca2+ loading of the SR.
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Phospholamban Function
PLB can be phosphorylated at Ser16 by cAMP-dependent protein kinase A (PKA),22 and at Thr17 by Ca2+-calmodulin–dependent kinase (CaMKII).23 Dephosphorylation of PLB is mediated by protein phosphatase-1 (PP1) and protein phosphatase-2a (PP2a).24 The expression of phospho-PKA was higher in TG myocytes, whereas PKA and phospho-CaMKII were similar in the 2 groups of cells. Moreover, the level of PP1 but not PP2a was decreased in TG myocytes (Figure 7A). Protein kinase C (PKC) increases PP1 activity that, in turn, attenuates PLB phosphorylation.25 PKC protein was increased in TG myocytes, but the protein amount does not necessarily reflect upregulation of kinase activity (Figure 7A and supplemental Figure III). Therefore, the enhanced function of phospho-PKA may account for the reduction in PP1 phosphatase activity, which was implicated in the phosphorylation of PLB and the potentiation of SERCA2a.
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Myofilament Proteins
TG myocytes had greater fractional shortening than WT myocytes in spite of similar Ca2+ transient amplitude. However, TG myocytes showed improved Ca2+ homeostasis, resulting in enhanced lusitropic function and shortening–frequency relationship and increased Ca2+ loading of the SR. In this regard, an increased myofilament Ca2+-binding affinity was documented by plotting cytosolic Ca2+ and sarcomere length during steady-state contraction. The terminal portion of these hysteresis loops (Figure 7B), when Ca2+ decays slowly, is indicative of Ca2+-binding affinity to the myofilaments. This relationship was shifted upwards in TG myocytes, pointing to increased sarcomere shortening for any given cytosolic Ca2+. These results support the notion that increases in myofilament Ca2+ responsiveness, together with an increased rate of SR Ca2+ uptake, promote a higher contractile function in TG myocytes. However, the lack of information on skinned fibers suggests caution in the interpretation of these results.
The state of phosphorylation of myosin light chain-2 (MLC-2) increases systolic function.26,27 Phospho–MLC-2 increases the force development of myocytes in response to intracellular [Ca2+]. This positive inotropic effect of phospho–MLC-2 appears to be linked to an increase in cross-bridge–cycling kinetics that, in turn, increases the amount of force generation at a given intracellular [Ca2+]. The protein level and state of phosphorylation of MLC-2 evaluated by Western blotting and immunocytochemistry were comparable in TG and WT myocytes (Figure 7C and 7D). Therefore, enhanced myocyte shortening in TG cannot be accounted for by changes in phospho–MLC-2.
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Discussion |
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The results of the current study indicate that the overexpression of Akt in myocyte nuclei had profound effects on the structure and function of the heart in the absence of cardiac hypertrophy. The ventricular myocardium was composed of a larger number of myocytes, which were smaller in size. Although myocardial mass and chamber volume were comparable in TG and WT, baseline ventricular hemodynamics was increased in TG and the enhanced cardiac performance involved both systolic and diastolic function. These positive changes at the organ level were sustained by corresponding positive changes in the mechanical behavior of ventricular myocytes. Peak shortening, velocity of shortening, velocity of relengthening and Ca2+ handling were all improved in TG myocytes. These parameters of potentiated myocyte contractility were paralleled by an increased activity of SERCA2a mediated by an increased phosphorylation of PLB. Together, these cellular adaptations associated with the nuclear localization of Akt in myocytes demonstrate that targeted expression of this serine–threonine kinase may have important clinical implications for the diseased heart.
Nuclear-Targeted Akt and the Heart Phenotype
Akt is a downstream effector molecule initiated by the activation of PI3K that is part of the signaling pathway modulated by the insulin-like growth factor (IGF)-1/IGF-1 receptor system or insulin.1 Phospho-Akt is translocated to the nucleus where it phosphorylates transcription factors for genes that oppose cell death and promote cell growth.8–11 The cytoplasmic overexpression of Akt in the heart leads to cardiac hypertrophy that is fully accounted for by an increase in myocyte volume without an increase in myocyte number.3–7,12 Conversely, the forced expression of IGF-1 results in an increase in the number of cardiomyocytes28 and skeletal muscle cells29 because IGF-1 is a powerful inducer of cell proliferation and survival. With time, IGF-1 is associated with an increase in myocyte size, although myocyte multiplication remains the predominant cellular response.28,29 Therefore, similarities exist in the role of cytoplasmic-targeted Akt and IGF-1 in cell viability, but Akt and IGF-1 have a substantially different impact on the pattern of myocyte growth.
An important finding of the current study is that nuclear-targeted Akt resulted in a cardiac phenotype, which was superior to that obtained with the cytoplasmic localization of Akt and the overexpression of IGF-1. The ventricular muscle mass was constituted by an increased number of myocytes, which were smaller in volume preventing the development of cardiac hypertrophy. This novel form of myocardial assembly was characterized by enhanced cardiac function measured echocardiographically and hemodynamically. The potentiation of cardiac performance in nuclear-targeted Akt mice was not observed with cardiac restricted IGF-1 overexpression28 or with the cytoplasmic localization of Akt.5–7,12
Mice with nuclear-targeted Akt have a 10-fold increase in total Akt protein, which is phosphorylated by PI3K at Thr308 and/or Ser473. Nuclear-targeted Akt enhances the nuclear levels of phospho-Akt at Ser473, whereas the cytoplasmic level of the phospho-protein is similar in WT and TG myocytes. In this regard, the infection of neonatal myocytes with an adenovirus carrying nuclear-targeted Akt does not result in an increased phosphorylation of cytoplasmic substrates of Akt, such as GSKß3 and Bad.8 Under this setting, Akt function is exerted almost exclusively at the level of the nucleus. This explains, at least in part, why the molecular consequences of Akt activation in myocytes are dramatically different when excess Akt activity is present in the cytoplasm5–7,12,30 as opposed to the nucleus.8 Cytoplasmic-targeted Akt phosphorylates GSKß3 that induces myocyte hypertrophy and interferes with the activation and commitment of cardiac progenitor cells.3,6 This is not the case in nuclear-targeted Akt,8 in which a significant increase in the generation of myocytes has been found by growth and differentiation of resident progenitor cells (M.S. and P.A., unpublished observations, 2005).
Nuclear-Targeted Akt and Myocyte Mechanics
Myocyte contraction and relaxation are under the control of the rise and decline in cytosolic Ca2+ levels.17 During the action potential, Ca2+ enters the cell via sarcolemmal L-type channels and ICaL triggers the release of Ca2+ from the SR by activating the RyR channels.31 The rise in intracellular free Ca2+ initiates contraction through the binding of Ca2+ to the myofilaments, whereas myocyte relaxation is promoted by the decrease in intracellular Ca2+ and dissociation of Ca2+ from the myofilaments. Lowering in cytosolic Ca2+ is accomplished mainly by Ca2+ sequestration into the SR by SERCA2a and Ca2+ extrusion from the cell by the NCX.32 In its phosphorylated form, PLB enhances SERCA2a and Ca2+ transport into the SR, regulating the rate of cardiac relaxation and the amount of Ca2+ stored in the SR.21
The ability of nuclear-targeted Akt to modify the mechanical behavior of myocytes in the absence of cellular hypertrophy has no precedent. The lack of changes in developed Ca2+, together with the increase in myofilament Ca2+ sensitivity, is consistent with the increase in fractional shortening and velocity of shortening and relengthening of TG myocytes. These properties differ somehow from the effects that IGF-1 overexpression has on myocyte contractility and myofilament Ca2+ sensitivity.33 Similarly, the consequences of nuclear-targeted Akt on myocyte performance markedly diverge from those associated with the cytoplasmic localization of this kinase.12 Conversely, nuclear-targeted Akt shows some of the properties of ß-adrenergic stimulation,17 which potentiates myocyte mechanics by enhancing PKA and, thereby, the phosphorylation of PLB. Activation of ß-receptors leads to myocyte apoptosis,34,35 whereas Akt protein has a potent antiapoptotic function. Because attenuation of cell death by ß-blockers has been implicated in the favorable outcome of heart failure in patients, nuclear-targeted Akt possesses all the beneficial consequences of enhanced myocyte contraction present with ß-adrenergic stimulation but, in contrast to ß1 receptor activation, has a powerful positive effect on myocyte survival.1,8
The improved myocyte contractility with nuclear-targeted Akt is largely related to the influx of Ca2+ into its site of storage with increased loading of the SR. The increase in SERCA2a activity by PLB phosphorylation may represent a potential mechanism for increased SR function in TG. Phosphatase PP1, which dephosphorylates PLB, is downregulated in TG myocytes, providing an additional pathway for an effective reuptake of Ca2+ by the SR. Depressed myocyte mechanics and abnormal intracellular Ca2+ cycling are typical features of the failing human heart. A decrease in PLB phosphorylation and an increase in expression and activity of NCX have been implicated in the defects of Ca2+ loading of the SR in human heart failure.21,32,36,37 Impaired SR function results in an elevation in Ca2+ concentration in the cytoplasm, which, in turn, leads to the formation of reactive oxygen species and the initiation of the endogenous cell death pathway.38,39 The nuclear localization of Akt operates against these negative cellular processes and support the notion that nuclear targeting of Akt may be a candidate strategy for cardiac failure.
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Acknowledgments |
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This work was supported by NIH grants HL-38132, AG-15756, HL-65577, HL-66923, HL-65573, HL33921, HL44659, AG-17042, AG-023071, HL-43023, HL-50142, AG-02617, and HL-081737, and by Fondazione Cassa di Risparmio Parma.
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Footnotes |
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Original received August 10, 2005; revision received November 3, 2005; accepted November 7, 2005.
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