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. 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. PKB/Akt, a 57-kDa protein, is a serine-threonine kinase and exists as three isoforms, α, β, and γ (Akt1, Akt2, and Akt3, respectively) (1Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar). Akt is activated by various growth stimuli including insulin, platelet-derived growth factor, insulin-like growth factor-1, and agonists for the β-adrenergic receptor, as well as stresses, such as shear stress (2Shiojima I. Walsh K. Circ. Res. 2002; 90: 1243-1250Crossref PubMed Scopus (854) Google Scholar). Activated Akt phosphorylates various downstream targets, thereby modulating diverse cellular functions including cell survival and apoptosis, protein synthesis, and glycogen metabolism (1Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar, 2Shiojima I. Walsh K. Circ. 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Chem. 2000; 275: 14466-14475Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 8Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Recent reports have shown that cardiac-specific overexpression of active mutants of Akt induces hypertrophy in transgenic mice in vivo (9Cook S.A. Matsui T. Li L. Rosenzweig A. J. Biol. Chem. 2002; 277: 22528-22533Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 10Shioi T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-2809Crossref PubMed Scopus (448) Google Scholar, 11Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12333-12338Crossref PubMed Scopus (404) Google Scholar). Akt activation also exerts a cardioprotective effect by inhibiting cardiomyocyte death during ischemia/reperfusion (12Mockridge J.W. Marber M.S. Heads R.J. Biochem. Biophys. Res. Commun. 2000; 270: 947-952Crossref PubMed Scopus (77) Google Scholar, 13Miao W. Luo Z. Kitsis R.N. Walsh K. J. Mol. Cell Cardiol. 2000; 32: 2397-2402Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 14Matsui T. Tao J. del Monte F. Lee K.H. Li L. Picard M. Force T.L. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 2001; 104: 330-335Crossref PubMed Scopus (597) Google Scholar, 15Matsui T. Li L. del Monte F. Fukui Y. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 1999; 100: 2373-2379Crossref PubMed Scopus (339) Google Scholar, 16Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (738) Google Scholar). The cardioprotection is associated with reduced infarct size as well as preserved function of surviving myocardium (14Matsui T. Tao J. del Monte F. Lee K.H. Li L. Picard M. Force T.L. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 2001; 104: 330-335Crossref PubMed Scopus (597) Google Scholar). 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 1The abbreviations used are: LVleft ventricularTGtransgenic mice with cardiac-specific overexpression of active AktSRsarcoplasmic reticulumSERCAsarcoplasmic reticulum Ca2+ ATPasePLBphospholambanNCXNa+/Ca2+ exchangerα1cL-type Ca2+ channelKIkinase inactiveWTwild type mice. dP/dtmax (11Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12333-12338Crossref PubMed Scopus (404) Google Scholar). 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 Ca2+ channel function and Ca2+-handling proteins, including sarcoplasmic reticulum (SR) Ca2+ ATPase 2a (SERCA2a), phospholamban (PLB), calsequestrin, the α-subunit of the L-type Ca2+ channel (α1c), and Na+/Ca2+ exchanger (NCX), as well as myocardial ryanodine receptor binding. Our results suggest that activation of Akt enhances Ca2+ transients and facilitates both contraction and relaxation in isolated cardiac myocytes, which is associated with enhanced Ca2+ influx and increased protein levels of SERCA2a. These features indicate a novel function of Akt in the mouse heart. left ventricular transgenic mice with cardiac-specific overexpression of active Akt sarcoplasmic reticulum sarcoplasmic reticulum Ca2+ ATPase phospholamban Na+/Ca2+ exchanger L-type Ca2+ channel kinase inactive wild type mice. Transgenic Mice—The generation of transgenic mice with cardiacspecific overexpression of constitutively active Akt (E40K Akt) (TG) has been described (11Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12333-12338Crossref PubMed Scopus (404) Google Scholar). We have also generated transgenic mice with 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 (37National Research Council Guide for the Care and Use of Laboratory Animals. National Research Council, National Academy Press, Washington, D. C.1996Google Scholar). Echocardiography—Mice were anesthetized with an intraperitoneal injection of ketamine (0.065 mg/g), acepromazine (0.02 mg/g), and xylazine (0.013 mg/g). The procedure for echocardiography has been reported previously (17Sadoshima J. Montagne O. Wang Q. Yang G. Warden J. Liu J. Takagi G. Karoor V. Hong C. Johnson G.L. Vatner D.E. Vatner S.F. J. Clin. Investig. 2002; 110: 271-279Crossref PubMed Scopus (151) Google Scholar, 18Asai K. Yang G.P. Geng Y.J. Takagi G. Bishop S. Ishikawa Y. Shannon R.P. Wagner T.E. Vatner D.E. Homcy C.J. Vatner S.F. J. Clin. Investig. 1999; 104: 551-558Crossref PubMed Scopus (106) Google Scholar). Measurement of Contraction and Relaxation and Ca2+Transients— Adult mouse ventricular cardiac myocytes were isolated as described previously (19Kim S.J. Yatani A. Vatner D.E. Yamamoto S. Ishikawa Y. Wagner T.E. Shannon R.P. Kim Y.K. Takagi G. Asai K. Homcy C.J. Vatner S.F. J. Clin. Investig. 1999; 103: 1089-1097Crossref PubMed Scopus (32) Google Scholar). In brief, the heart was rapidly excised and submerged in Ca2+-free Tyrode's solution containing (mmol/liter): 140 NaCl, 5.4 and The heart was and ± were with and during were and contraction was a as described previously (19Kim S.J. Yatani A. Vatner D.E. Yamamoto S. Ishikawa Y. Wagner T.E. Shannon R.P. Kim Y.K. Takagi G. Asai K. Homcy C.J. Vatner S.F. J. Clin. Investig. 1999; 103: 1089-1097Crossref PubMed Scopus (32) Google Scholar). The solution The contractile were from contraction and the rate of Relaxation the rate of the for were also Ca2+ function was examined in to thapsigargin and in both WT and were with Fura-2 and Ca2+ transients were the (19Kim S.J. Yatani A. Vatner D.E. Yamamoto S. Ishikawa Y. Wagner T.E. Shannon R.P. Kim Y.K. Takagi G. Asai K. Homcy C.J. Vatner S.F. J. Clin. Investig. 1999; 103: 1089-1097Crossref PubMed Scopus (32) Google Scholar). L-type Ca2+ were as described previously (19Kim S.J. Yatani A. Vatner D.E. Yamamoto S. Ishikawa Y. Wagner T.E. Shannon R.P. Kim Y.K. Takagi G. Asai K. Homcy C.J. Vatner S.F. J. Clin. Investig. 1999; 103: 1089-1097Crossref PubMed Scopus (32) Google Scholar). The solution (mmol/liter): and The solution (mmol/liter): and These of Ca2+ from other and from the Na+/Ca2+ for and ventricular was and in the of the were in NaCl, with and kinase of protein were in by and for SERCA2a and were for with a of from or mouse in containing and for were with a of cardiac in the for were with a of in and for were with a of in and of the were by a with and for protein were with a of for and with of for was and the were by SERCA2a protein was for as described receptor were as described previously D.E. N. K. Shannon R.P. Vatner S.F. Circulation. 90: PubMed Scopus Google Scholar). were of to and of protein in 0.3 in a of ryanodine was used to determine binding. was for were in and by with of were in of and in a for were by the protein were the was the versus by the mouse heart was in and The was in a solution containing in the of of as a and was isolated by the in a for the was and the was in was to the solution to a of and the was with The was in and in was from of by and to the was in a of 50 in the of of The were in a for of as described The and and were used for and of for for for M.J. Biochem. J. 2001; PubMed Google and of for for for (17Sadoshima J. Montagne O. Wang Q. Yang G. Warden J. Liu J. Takagi G. Karoor V. Hong C. Johnson G.L. Vatner D.E. Vatner S.F. J. Clin. Investig. 2002; 110: 271-279Crossref PubMed Scopus (151) Google Scholar). The were and by of Adult and rat cardiac myocyte were as described previously S.J. Yatani A. Kim Y.K. Takagi G. R. Vatner D.E. Vatner S.F. Circ. Res. 2001; PubMed Scopus Google to determine overexpression of Akt enhances the expression of SERCA protein as a mechanism responsible for the enhanced myocyte contractile and relaxation function. either Akt of or of was myocyte for have been described (7Morisco C. Zebrowski D. Condorelli G. Tsichlis P. Vatner S.F. Sadoshima J. J. Biol. Chem. 2000; 275: 14466-14475Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). as ± S.E. of the the was by of or by and was p < The myocyte were for for weight was increased (p < 0.01) in TG ± compared with WT ± mg/g). The results of in I. rate was TG and WT. The was increased (p < 0.01) in TG ± compared with WT ± were were reduced by in TG compared with WT (p < 0.05). The LV ejection fraction was enhanced (p < 0.01) in TG (84 ± compared with WT mice (73 ± 1%). These results suggest that TG have hypertrophy with increased ventricular function by rate ± ± ± ± ± ± < versus WT. ± ± ± < versus WT. ± fraction ± ± < versus WT. ± ± ± < versus WT. ± p < versus WT. ± S.E. in a Contraction and and Ca2+ in myocytes isolated from WT and from in that contraction was increased in TG myocytes compared with WT myocytes (6.1 ± 0.2 versus 3.5 ± 0.2%, p < and the rate of contraction was also increased in TG myocytes compared with WT myocytes ± 15 versus ± μm/s, p < 0.01). Relaxation function in TG myocytes was also enhanced compared with WT the rate of (+dL/dt) was (214 ± 15 versus 98 ± 18 μm/s, p < 0.01) and the for relaxation was in TG myocytes ± versus ± p < 0.01). The effect of constitutively active Akt overexpression is the kinase of Akt, myocytes isolated from Tg-KI-Akt did not show enhanced contraction and relaxation function. These results suggest that increased myocardial contraction in TG in vivo is also observed in cardiac myocytes isolated from relaxation, and in isolated ventricular cardiac ± ± < versus WT. ± ± 0.2 ± ± < versus WT. ± ± ± ± < versus WT. ± ± ± ± < versus WT. ± ± of ± ± < versus WT. ± ± ± ± < versus WT. ± ± 50 ± ± < versus WT. ± ± ± ± < versus WT. ± ± of p < versus WT. ± S.E. in a To the enhanced contraction and relaxation in TG myocytes by in Ca2+ we Ca2+ shown in and the of Ca2+ transients in TG myocytes was increased (396 ± 50 versus 250 ± 24 nmol/liter, p < 0.05). were in levels of Ca2+ ± versus ± nmol/liter, p not The for Ca2+ was also in TG myocytes compared with WT myocytes ± versus ± p < 0.05). Thus, both Ca2+ levels and Ca2+ increased in myocytes isolated from isolated from Tg-KI-Akt did not show in Ca2+ levels and Ca2+ These results indicate that in contractile function and Ca2+ transients Akt L-type Ca2+ influx L-type Ca2+ plays an role in we examined L-type Ca2+ (ICa) in LV myocytes isolated from TG and WT LV myocyte size by cell was in TG myocytes compared with WT myocytes with the heart weight The in show from TG and WT myocytes. In both was activated and However, the by the cell was (p < 0.01) in TG myocytes ± 0.3 compared with WT myocytes ± 0.3 The kinetics of was in TG myocytes compared with WT myocytes In mouse ventricular myocytes, is by Ca2+ in the (19Kim S.J. Yatani A. Vatner D.E. Yamamoto S. Ishikawa Y. Wagner T.E. Shannon R.P. Kim Y.K. Takagi G. Asai K. Homcy C.J. Vatner S.F. J. Clin. Investig. 1999; 103: 1089-1097Crossref PubMed Scopus (32) Google Scholar, H. Y. Luo W. Yatani A. J. 1997; Google Scholar, A. K. H. J. Mol. Cell Cardiol. 1999; Full Text PDF PubMed Scopus Google Scholar). To the in rate WT and TG myocytes was to the of Ca2+ from the the Ca2+ was by ryanodine the of the rate of as by was increased in both WT and TG myocytes, and in the rate was ± versus ± The results indicate that the observed in TG myocytes be to enhanced Ca2+ and both enhanced and relaxation of the Ca2+ be by in Ca2+-handling proteins, the expression of these was by The protein expression of Akt in myocardium was increased in TG compared with WT mice. SERCA2a protein levels were increased (p < 0.05) by 6.6-fold in TG compared with WT mice. In protein levels of calsequestrin, and were not TG and WT mice and The and of ryanodine were WT and TG versus versus in WT and TG that was in SERCA2a levels TG and WT Thus, the increase in SERCA2a protein in TG of Akt of SERCA2a in Adult determine overexpression of Akt is to increase SERCA2a protein expression in cardiac myocytes, we adenovirus-mediated overexpression of Akt in cultured adult rat ventricular cardiac myocytes. overexpression of Akt was by of the with that expression was enhanced by p < 0.05) compared with cardiac myocytes SERCA2a was in in myocytes, only a a of was in Akt myocytes. In the with the that a of SERCA2a not Up-regulation of SERCA2a and Relaxation in from examined the effect of a of contraction of isolated myocytes. The of thapsigargin inhibiting contraction by was in myocytes isolated from TG than in from WT relaxation function in TG myocytes was also thapsigargin These results with the that enhanced SERCA2a plays an important role in mediating increased cardiac myocyte contraction and relaxation in Akt plays a role in cell apoptosis, and protein B.A. Biochem. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We that overexpression of constitutively active Akt in the mouse heart increased both LV ejection in and isolated myocyte contraction in study the TG demonstrated increased LV with a increase in LV ejection fraction (11Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12333-12338Crossref PubMed Scopus (404) Google Scholar). The to increased LV ejection fraction in the study (11Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12333-12338Crossref PubMed Scopus (404) Google be to heart which was in TG than the heart were in the in the the LV function in the of LV which is to LV function. The of the study to constitutively active Akt has contractility and Ca2+ in isolated cardiac and to the downstream mechanism of the enhanced cardiac myocyte function by Akt We have demonstrated that the enhanced cardiac function in TG in vivo is associated with increased intrinsic contractile and relaxation function in isolated in the increased contraction and relaxation in myocytes isolated from TG the increased and more in Ca2+ The in isolated myocytes also demonstrated increased Ca2+ channel currents. These in isolated myocytes were not observed in Tg-KI-Akt, that the kinase of Akt. that activation of Akt contraction and relaxation in Ca2+ in cardiac myocytes. be that the enhanced cardiac contractility was not observed in TG mice with cardiac-specific overexpression of constitutively active an of Akt T. Kang P.M. Douglas P.S. J. C.M. J. Cantley L.C. Izumo S. J. 2000; PubMed Scopus Google or in other forms of constitutively active Akt and T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-2809Crossref PubMed Scopus (448) Google Scholar, T. Li L. J.C. S.A. T. Picard M.H. R. Rosenzweig A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google from the used in this study the that mice cardiac hypertrophy. Furthermore, activated by of and negatively cardiac contractility M. G. I. R. H. T. E. A. T. J. R. H. V. G. L. A. J. C. Izumo S. S. M. P. J. 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E. J. Clin. Investig. 1997; 100: PubMed Scopus Google Scholar). In the study, SERCA2a protein levels were in TG mice. we that of thapsigargin to contraction in cardiac myocytes isolated from TG and the enhanced relaxation function in TG myocytes was in the of These results suggest that increased levels of SERCA2a to enhanced Ca2+ transients and contraction as well as relaxation in cardiac myocytes. Our results indicate that of SERCA2a expression by Akt is by The of mouse SERCA2a by Akt. Our cultured adult ventricular cardiac myocytes that adenovirus-mediated overexpression of Akt increased expression of SERCA2a. Furthermore, the only a of SERCA2a Akt and only the was observed not with the that SERCA2a is Akt is SERCA2a is a of Akt, and activation of Akt enhances or of remains to be We and have shown that activation of Akt cardiac myocyte hypertrophy both in vitro and in vivo (7Morisco C. Zebrowski D. Condorelli G. Tsichlis P. Vatner S.F. Sadoshima J. J. Biol. 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Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (738) Google of Akt be an of the contractility of the heart and Ca2+ in to In transgenic overexpression of protein kinase Akt enhances myocyte contractility and relaxation of Ca2+ The cellular of L-type Ca2+ channel function and of SERCA2a.