Metabolic Switch and Hypertrophy of Cardiomyocytes following Treatment with Angiotensin II Are Prevented by AMP-activated Protein Kinase

Angiotensin II induces cardiomyocyte hypertrophy, but its consequences on cardiomyocyte metabolism and energy supply are not completely understood. Here we investigate the effect of angiotensin II on glucose and fatty acid utilization and the modifying role of AMP-activated protein kinase (AMPK), a key regulator of metabolism and proliferation. Treatment of H9C2 cardiomyocytes with angiotensin II (Ang II, 1 μm, 4 h) increased [3H]leucine incorporation, up-regulated the mRNA expression of the hypertrophy marker genes MLC, ANF, BNP, and β-MHC, and decreased the phosphorylation of the negative mTOR-regulator tuberin (TSC-2). Rat neonatal cardiomyocytes showed similar results. Western blot analysis revealed a time- and concentration-dependent down-regulation of AMPK-phosphorylation in the presence of angiotensin II, whereas the protein expression of the catalytic α-subunit remained unchanged. This was paralleled by membrane translocation of glucose-transporter type 4 (GLUT4), increased uptake of [3H]glucose and transient down-regulation of phosphorylation of acetyl-CoA carboxylase (ACC), whereas fatty acid uptake remained unchanged. Similarly, short-term transaortic constriction in mice resulted in down-regulation of P-AMPK and P-ACC but up-regulation of GLUT4 membrane translocation in the heart. Preincubation of cardiomyocytes with the AMPK stimulator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 1 mm, 4 h) completely prevented the angiotensin II-induced cardiomyocytes hypertrophy. In addition, AICAR reversed the metabolic effects of angiotensin II: GLUT4 translocation was reduced, but ACC phosphorylation and TSC phosphorylation were elevated. In summary, angiotensin II-induced hypertrophy of cardiomyocytes is accompanied by decreased activation of AMPK, increased glucose uptake, and decreased mTOR inhibition. Stimulation with the AMPK activator AICAR reverses these metabolic changes, increases fatty acid utilization, and inhibits cardiomyocyte hypertrophy. Angiotensin II induces cardiomyocyte hypertrophy, but its consequences on cardiomyocyte metabolism and energy supply are not completely understood. Here we investigate the effect of angiotensin II on glucose and fatty acid utilization and the modifying role of AMP-activated protein kinase (AMPK), a key regulator of metabolism and proliferation. Treatment of H9C2 cardiomyocytes with angiotensin II (Ang II, 1 μm, 4 h) increased [3H]leucine incorporation, up-regulated the mRNA expression of the hypertrophy marker genes MLC, ANF, BNP, and β-MHC, and decreased the phosphorylation of the negative mTOR-regulator tuberin (TSC-2). Rat neonatal cardiomyocytes showed similar results. Western blot analysis revealed a time- and concentration-dependent down-regulation of AMPK-phosphorylation in the presence of angiotensin II, whereas the protein expression of the catalytic α-subunit remained unchanged. This was paralleled by membrane translocation of glucose-transporter type 4 (GLUT4), increased uptake of [3H]glucose and transient down-regulation of phosphorylation of acetyl-CoA carboxylase (ACC), whereas fatty acid uptake remained unchanged. Similarly, short-term transaortic constriction in mice resulted in down-regulation of P-AMPK and P-ACC but up-regulation of GLUT4 membrane translocation in the heart. Preincubation of cardiomyocytes with the AMPK stimulator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 1 mm, 4 h) completely prevented the angiotensin II-induced cardiomyocytes hypertrophy. In addition, AICAR reversed the metabolic effects of angiotensin II: GLUT4 translocation was reduced, but ACC phosphorylation and TSC phosphorylation were elevated. In summary, angiotensin II-induced hypertrophy of cardiomyocytes is accompanied by decreased activation of AMPK, increased glucose uptake, and decreased mTOR inhibition. Stimulation with the AMPK activator AICAR reverses these metabolic changes, increases fatty acid utilization, and inhibits cardiomyocyte hypertrophy. Left ventricular (LV) 3The abbreviations used are: LV, left ventricular; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK, AMP-activated protein kinase; Ang II, angiotensin II; GLUT4, glucose transporter 4; PBS, phosphate-buffered saline; TAC, transaortic constriction; TSC-2, tuberous sclerosis complex 2; MLC, myosin light chain; ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain. hypertrophy is a major independent risk factor for premature death (1Bayes-Genis A. Guindo J. Vinolas X. Tomas L. Elosua R. Duran I. Bayes D.L. Am. J. Cardiol. 1995; 76: 54D-59DAbstract Full Text PDF PubMed Scopus (24) Google Scholar). Myocardial hypertrophy is a response to hemodynamic overload such as hypertension or aortic valve stenosis. A fundamental mechanism leading to both hypertrophy and subsequent heart failure is activation of the renin-angiotensin-aldosteron system. All components of the system are expressed at the mRNA and protein levels in the heart (2Suzuki J. Matsubara H. Urakami M. Inada M. Circ. Res. 1993; 73: 439-447Crossref PubMed Scopus (274) Google Scholar), circulating plasma concentrations of angiotensin II (Ang II) correlate to the extend of LV hypertrophy (3Weber K.T. Brilla C.G. Campbell S.E. Guarda E. Zhou G. Sriram K. Basic Res. Cardiol. 1993; 88: 107-124PubMed Google Scholar) and the progression to heart failure (4Neri Serneri G.G. Boddi M. Modesti P.A. Cecioni I. Coppo M. Padeletti L. Michelucci A. Colella A. Galanti G. Circ. Res. 2001; 89: 977-982Crossref PubMed Scopus (153) Google Scholar), and the increase in left ventricular mass can be prevented with an angiotensin converting enzyme inhibitor or angiotensin receptor blocker (5Baker K.M. Chernin M.I. Wixson S.K. Aceto J.F. Am. J. Physiol. 1990; 259: H324-H332PubMed Google Scholar). Molecular mechanisms by which the renin-angiotensin-aldosteron system evokes cardiac hypertrophy are activation of protein kinase cascades, initiation of a fetal-like gene program, impaired calcium handling, and increased production of reactive oxygen species (6Custodis F. Eberl M. Kilter H. Bohm M. Laufs U. Cardiovasc. Res. 2006; 71: 342-351Crossref PubMed Scopus (77) Google Scholar). Changes in substrate utilization occurring during cardiomyocyte hypertrophy are not well known, and their consequences on cardiomyocyte metabolism and energy supply are not completely understood. A shift in the substrate pattern from fatty acid oxidation to glucose utilization appears to occur in different forms of LV hypertrophy (7Sambandam N. Lopaschuk G.D. Brownsey R.W. Allard M.F. Heart Fail. Rev. 2002; 7: 161-173Crossref PubMed Scopus (138) Google Scholar), but whether metabolic changes precede hypertrophy or result from it and which pathways are involved is unknown. A key metabolic regulator is AMP-activated protein kinase (AMPK), a heterotrimeric protein that is centrally involved in controlling cellular energy homeostasis. Its activation through metabolic stress stimulates energy-generating processes and has been shown to regulate hypertrophy-regulating genes in several models (8Motoshima H. Goldstein B.J. Igata M. Araki E. J. Physiol. 2006; 574: 63-71Crossref PubMed Scopus (445) Google Scholar). When AMPK binds AMP the α-subunit gets sensitized to phosphorylation by upstream kinases and increases kinase activity. Upon activation AMPK increases fatty acid oxidation by phosphorylating ACC, in turn decreasing malonyl-CoA content and activating carnitine palmitoyltransferase-I. In several tissues AMPK has been shown to also regulate glucose metabolism (9Minokoshi Y. Alquier T. Furukawa N. Kim Y.B. Lee A. Xue B. Mu J. Foufelle F. Ferre P. Birnbaum M.J. Stuck B.J. Kahn B.B. Nature. 2004; 428: 569-574Crossref PubMed Scopus (1348) Google Scholar) and translocation of glucose transporter 4 (GLUT4) to the plasma membrane (10Yamaguchi S. Katahira H. Ozawa S. Nakamichi Y. Tanaka T. Shimoyama T. Takahashi K. Yoshimoto K. Imaizumi M.O. Nagamatsu S. Ishida H. Am. J. Physiol. 2005; 289: E643-E649Crossref PubMed Scopus (114) Google Scholar). Activated AMPK mediates changes in protein expression through regulating the mTOR pathway in multiple cell types by increasing phosphorylation of tuberin (TSC-2) (8Motoshima H. Goldstein B.J. Igata M. Araki E. J. Physiol. 2006; 574: 63-71Crossref PubMed Scopus (445) Google Scholar). mTOR is known to be crucial in the development and progression of LV hypertrophy (11McMullen J.R. Sherwood M.C. Tarnavski O. Zhang L. Dorfman A.L. Shioi T. Izumo S. Circulation. 2004; 109: 3050-3055Crossref PubMed Scopus (410) Google Scholar). But the relevance of AMPK activation in cardiomyocyte hypertrophy and metabolism is still in question. Therefore, the aim of the study was to examine the effects of angiotensin II on glucose and fatty acid utilization in cardiomyocytes, and whether metabolic alterations through modulation of AMP-activated protein kinase would prevent angiotensin II-induced hypertrophy. Cell Culture—Rat neonatal cardiomyocytes were isolated as described (12Laufs U. Kilter H. Konkol C. Wassmann S. Bohm M. Nickenig G. Cardiovasc. Res. 2002; 53: 911-920Crossref PubMed Scopus (189) Google Scholar). Briefly, hearts of neonatal rats 2–4 days of age were digested with pankreatin and collagenase type 2, the cell suspension was centrifuged and filtered to extract fibroblasts, and cardiomyocytes were counted and seeded at 2–4 × 106 per well. After overnight incubation viability and contractile function was evaluated microscopically, cardiomyocytes were washed and serum starved before using for experiments. H9C2 rat cardiomyocytes (LGC Promochem, Wesel, Germany) are an immortalized cell line derived from embryonic rat hearts using selective serial passage. Cells were grown on tissue culture dishes in Dulbecco's modified Eagle's medium complete cell culture medium (Invitrogen), supplemented with 10% fetal calf serum, 1% penicillin and streptomycin, maintained in 95% air and 5% CO2 at 37 °C. Cells were not allowed to become confluent to prevent the loss of differentiation potential. Experiments were performed at passage 15–23. For protein and mRNA extraction cells were seeded in 10-cm dishes, starved for 24 h in Dulbecco's modified Eagle's medium starvation medium and used for experiments. Cells used for metabolic uptake assays were seeded in triplicates in six-well plates for 48 h, washed twice with 2 ml of PBS, and starved for 24 h. After starvation the cells were washed once with PBS and used for experiments. Treatment Conditions—For angiotensin II treatment cells were incubated with 1 μm angiotensin II (purchased from Sigma) for the indicated time periods (5 min to 24 h). AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) treatment was done in a concentration of 1 mm for 2 h (from Sigma). In the case of combined incubation AICAR pre-treatment was done for 2 h with 1 mm, followed by angiotensin treatment for 4 h with 1 μm. Finally cells were washed with 2 ml of PBS, lysed with 200 μl of ice-cold lysis buffer, scraped, and homogenized with 5 min ultrasound. After homogenization debris was removed by centrifugation at 14,000 × g for 15 min at 4 °C and supernatant was transferred to Eppendorf cups for storage at −20 °C. Western Analysis—For Western blot analysis protein concentration in cell lysates was measured using a Bio-Rad DC Protein Assay Kit. Protein (50 μg) was loaded in each slot of a 10% polyacrylamide gel, separated at 120 V for 2 h and transferred with 350 mA for 1.5 h. Membrane were stained with Ponceau solution, washed with TBS-Tween, and blocked with 5% milk for 30 min at 4 °C. Incubations with primary antibodies were done at 4 °C for 12 h. Primary antibodies were purchased from New England Biolabs (P-AMPK, P-ACC) and Santa Cruz Biotechnology (GLUT4, α2-AMPK, glyceraldehyde-3-phosphate dehydrogenase, P-tuberin). Secondary antibodies were purchased from Santa Cruz and incubated at room temperature for 30 min (goat) and 90 min (rabbit), respectively. After washing with PBS-Tween (20 min, twice) and PBS (10 min, twice) membranes were developed with ECL scintillation fluid obtained from GE Healthcare and exposed to hyperfilm. The amount of GLUT4 in the membrane fraction was determined after membrane preparation. Cells were harvested as described above, mixed at equal volumes with 1 mm KCl, and centrifuged for 1 h at 100,000 × g at 4 °C. The resultant supernatant was collected as cytosolic fraction. The pellet was resuspended, centrifuged at 100,000 × g for 1 h, and the resulting supernatant regarded as the membrane fraction. Separation of cytosolic and membrane proteins was controlled by Western blotting using glyceraldehyde-3-phosphate dehydrogenase. Bands were scanned using XnView normalized for glyceraldehyde-3-phosphate dehydrogenase and the density values were expressed as % of control. Real-time Reverse Transcriptase-PCR—For reverse transcriptase-PCR RNA was extracted by the chloroform/isopropyl alcohol method. After photometric quantification of the isolated RNA, reverse transcriptase-PCR was performed with the TaqMan System (13Laufs U. Werner N. Link A. Endres M. Wassmann S. Jurgens K. Miche E. Bohm M. Nickenig G. Circulation. 2004; 109: 220-226Crossref PubMed Scopus (755) Google Scholar). mRNA expression was normalized to expression of the housekeeping gene 18S. The primers were as follows: for ANF, 5-TGG GCT CCT TCT CCA TCA CC and 5-GCC AAA AGG CCA GGA AGA GG; for BNP, 5-CAG AAC AAT CCA CGA TGC AG and 5-GCT GTC TCT GAG CCA TTT CC; for β-MHC, 5-GCC TAC CTC ATG GGA CTG AA and 5-ACA TTC TGC CCT TTG GTG AC; for MLC, 5-AGG CCT TCA CAA TCA TGG AC and 5-TCG TTT TTC ACG TTC ACT CG; and for CTG TTT and GAG ACT A. of acid [3H]leucine was as a of protein in cardiac Assay was performed as described (6Custodis F. Eberl M. Kilter H. Bohm M. Laufs U. Cardiovasc. Res. 2006; 71: 342-351Crossref PubMed Scopus (77) Google Scholar). Cells were incubated with the indicated treatment (Ang II for AICAR for 2 AICAR for 4 and 1 was for 12 h at 37 cells were washed with PBS, and harvested in ice-cold lysis were collected for scintillation glucose uptake cardiomyocytes were or with Ang II AICAR of AICAR Ang II for min, followed by the of for an 30 min at 37 °C. was by the washing the cells with PBS, and in 1 ml of lysis were for uptake as per min as by scintillation uptake was expressed as a of uptake of cardiomyocytes were or with Ang II AICAR or AICAR Ang II for was by 1 of incubation at 37 °C for 30 min, and by washing with 1 ml of ice-cold Cells were with 1 ml of lysis and and was determined by scintillation cellular fatty acid cardiomyocytes were seeded on cell culture in plates and with 1 μm angiotensin II for 4 h, 1 mm AICAR for 2 h. Cells were washed twice with μl of ice-cold PBS and with The were in of for min at washed with to the followed by a 1% acid in and was done by a using a and analysis with were by the of the and with of and in with mice Germany) were were with for transaortic constriction O. G. F. Bohm M. Laufs U. J. Am. Cardiol. PubMed Scopus Google Scholar). After mice were by a on The was in the and aortic constriction was performed by a a to a mm in and a aortic constriction of A was used for LV was as an increase in left ventricular to at All mice developed an increase in left ventricular of mice a and showed increase in or After 30 min of LV hearts were and in were by All values are expressed as S.E. and analysis of for multiple were were performed with the was were at Angiotensin II-induced Changes in TSC and mRNA of in H9C2 of H9C2 cells with angiotensin II (Ang 1 μm for 4 h, an increase in to with 4 per mRNA expression of the fetal genes atrial natriuretic factor brain natriuretic β-myosin heavy and myosin light to the housekeeping gene was increased to and 5 for MLC, 4 for BNP, of the negative mTOR regulator tuberin was decreased to per Rat neonatal cardiomyocytes showed similar effects not Angiotensin II GLUT4 and in we determined the changes in substrate Treatment with Ang II μm, 4 h) increased the fraction of GLUT4 in rat neonatal cardiomyocytes to accompanied by an increase in cellular uptake of to Angiotensin II ACC in of H9C2 cardiomyocytes to angiotensin II for 2 h decreased the phosphorylation of acetyl-CoA carboxylase to per This effect was after 4 h. Angiotensin μm for 4 h) not the uptake of acid Angiotensin II of AMP-activated Protein a of AMPK we the phosphorylation of key metabolic Treatment with angiotensin II to a of AMPK phosphorylation in a time- and concentration-dependent The effect of was with 1 μm Ang II at an incubation of 4 h, of AMPK by the remained 4 for Ang II) Preincubation with the AICAR mm, 1 h) completely reversed the angiotensin II-induced of AMPK, 4 Ang II reverses angiotensin II-induced AMPK inhibition. Preincubation with the AMPK activator AICAR mm, 1 h) reverses the angiotensin II-induced of AMPK expression of the catalytic 4 Ang II glyceraldehyde-3-phosphate of AMPK Stimulation on whether these changes in AMPK phosphorylation the shift in glucose utilization, we performed the metabolic treatment with the AMPK activator mm, 4 h) and after followed by angiotensin II treatment μm, 4 h) 4 Ang II a in GLUT4 translocation to with AICAR treatment and after AICAR with Ang II remained after AICAR pre-treatment and AICAR treatment as well. In ACC phosphorylation and fatty acid uptake were increased by AMPK activation to and an effect that was not reversed by angiotensin II Angiotensin II but reversed the activation of acid uptake to of Ang II of Angiotensin II and AICAR Treatment on the consequences of a of fatty acid uptake and fatty acid oxidation Ang II and AICAR was Treatment with Ang II μm, 4 h) a increase of fatty to which was prevented by pre-treatment with the AMPK activator AICAR 4 Ang II of AMPK and GLUT4 the cardiac of AMPK, and GLUT4 in TAC was to increase cardiac in mice 4 O. G. F. Bohm M. Laufs U. J. Am. Cardiol. PubMed Scopus Google Scholar). with 30 min after TAC the hearts showed a down-regulation of P-AMPK to This was with a of P-ACC to and up-regulation of GLUT4 membrane translocation to of by Stimulation of AMPK and of the of the substrate changes on cardiomyocyte protein and of hypertrophy was of AMPK by AICAR mm, 4 h) a increase in tuberin phosphorylation to per of mTOR The mRNA expression of ANF, BNP, and was to and 37 5% with per Angiotensin II-induced [3H]leucine was prevented by activation of AMPK 12 Ang II AICAR that angiotensin II-induced hypertrophy in cardiomyocytes is accompanied by changes in substrate The phosphorylation of AMP-activated protein kinase as a metabolic fatty acid that is to prevent cardiomyocyte hypertrophy. is that substrate utilization a crucial role in cardiac contractile and in to the heart N. Rev. Physiol. PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar) shown that after min of the phosphorylation of ACC was increased AMPK phosphorylation was whereas in the ACC phosphorylation was whereas AMPK phosphorylation was metabolic appears to be in different cell and different be for to cell culture in models and Here we the cell culture in a similar a short-term increase of in mice heart. these to be in the of the role of AMPK, we cardiomyocytes with the AMPK activator AICAR or before the of hypertrophy with angiotensin AMPK activation a metabolic utilization of fatty glucose AMPK activation a increase of the phosphorylation of ACC that was not by angiotensin II This was accompanied by an increase in acid uptake, both an increased uptake and of This effect was reversed by Ang II a that has to be glucose metabolism we a in GLUT4 translocation AICAR whereas glucose uptake remained with The in glucose uptake is to the expression of glucose in the cardiomyocyte plasma the cellular J. 1993; PubMed Scopus Google Scholar). the metabolic effects of AMPK activation in cardiomyocytes were paralleled by effects on protein and expression of substrate utilization as a key hypertrophy regulator in cardiac and the metabolism and hypertrophy. that angiotensin II-induced hypertrophy of cardiomyocytes on the of AMPK and changes in substrate utilization to the of glucose Stimulation of fatty acid oxidation by AMPK activation cardiomyocyte hypertrophy and a these are of in has to be in for