Akt1–Mediated Skeletal Muscle Growth Attenuates Cardiac Dysfunction and Remodeling After Experimental
Myocardial Infarction
Satoshi Araki,MD;Yasuhiro Izumiya,MD,PhD;Shinsuke Hanatani,MD;Taku Rokutanda,MD;
Hiroki Usuku,MD,PhD;Yuichi Akasaki,MD,PhD;Toru Takeo,PhD;Naomi Nakagata,PhD;
Kenneth Walsh,PhD;Hisao Ogawa,MD,PhD
Background—It is appreciated that aerobic endurance exerci can attenuate unfavorable myocardial remodeling following myocardial infarction.In contrast,little is known about the effects of increasing skeletal muscle mass,typically achieved through resistance training,on this process.Here,we utilized transgenic(TG)mice that can induce the growth of functional skeletal muscle by switching Akt1signaling in muscle fibers to asss the impact of glycolytic muscle growth on post-myocardial infarction cardiac remodeling.
Methods and Results—Male-noninduced TG mice and their nontransgenic littermates(control)were subjected to left anterior coronary artery ligation.Two days after surgery,mice were provided doxycyclin
e in their drinking water to activate Akt1 transgene expression in a skeletal muscle-specific manner.Myogenic Akt1activation led to diminished left ventricular dilation and reduced contractile dysfunction compared with control mice.Improved cardiac function in Akt1TG mice was coupled to diminished myocyte hypertrophy,decread interstitial fibrosis,and incread capillary density.ELISA and protein array analys demonstrated that rum levels of proangiogenic growth factors were upregulated in Akt1TG mice compared with control mice.Cardiac eNOS was activated in Akt1TG mice after myocardial infarction.The protective effect of skeletal muscle Akt activation on cardiac remodeling and systolic function was abolished by treatment with the eNOS inhibitor L-NAME. Conclusions—Akt1–mediated skeletal muscle growth attenuates cardiac remodeling after myocardial infarction and is associated with an incread capillary density in the heart.This improvement appears to be mediated by skeletal muscle to cardiac communication,leading to activation of eNOS-signaling in the heart.(Circ Heart Fail.2012;5:116-125.) Key Words:angiogenesisⅢexerci trainingⅢmyocardial infarctionⅢnitric oxide syntha
Ⅲremodeling heart failure
L oss of skeletal muscle mass is frequently obrved in patients with endstage chronic heart failure(CHF).It is well-recognized that skeletal muscle wasting is a strong independent risk factor for
中国邮政银行个人网上银行
mortality in the patient popula-tions.1A good correlation between lean muscle mass and exerci capacity has been found in patients with CHF.2 Therefore,maintaining or increasing skeletal muscle mass could be beneficial in patients with CHF.
Clinical Perspective on p125 Conventional endurance training leads to an increa in the proportion of slow/oxidative fibers,which are high in mito-chondrial content.3This type of exerci training elicits a variety of metabolic and morphological respons in skeletal muscle,including mitochondrial biogenesis.4Multiple lines of clinical evidence indicate that decread percentage of oxidative fibers,mitochondrial dysfunction,and vascular rarefaction are prominent features of skeletal muscle in patients with CHF.5It is widely held that aerobic exerci training improves functional capacity and prognosis in pa-tients with CHF.6Therefore,endurance training intended to increa oxidative fiber function is recognized as rational treatment strategy for CHF patients.7
Although high intensity treadmill exerci can have a modest effect in promoting muscle growth,8lean muscle mass is typically developed through resistance exerci training. Resistance exerci training is predominantly associated with an increa in protein synthesis and hypertrophy of fast/
Received March15,2011;accepted November28,2011.
From the Department of Cardiovascular Medicine(S.A.,Y.I.,S.H.,T.R.,H.U.,H.O.),Faculty of Life Sciences,Kumamoto University,Kumamoto, Japan;Molecular Cardiology(Y.A.,K.W.),Whitaker Cardiovascular Institute,Boston University School of Medicine,Boston,MA;Division of Reproductive Engineering(T.T.,N.N.),Center for Animal Resources and Development,Kumamoto University,Kumamoto,Japan.
Guest Editor for this article was Douglas L.Mann,MD.
The online-only Data Supplement is available with this article at circheartfailure.ahajournals/lookup/suppl/doi:10.1161/ CIRCHEARTFAILURE.111.964783/-/DC1.
Correspondence to Yasuhiro Izumiya,MD,PhD,Department of Cardiovascular Medicine,Faculty of Life Sciences,Kumamoto University,1-1-1 Honjo,Kumamoto,Kumamoto860-8556,Japan.E-mail izumiya@kumamoto-u.ac.jp
©2011American Heart Association,Inc.
Circ Heart Fail is available at circheartfailure.ahajournals DOI:10.1161/CIRCHEARTFAILURE.111.964783
glycolytic fibers.9This type of exerci promotes gains in maximal force output but has minimal effects on muscle fiber phenotype transformation.10Recent work has shown that incread skeletal muscle mass and glycolytic capacity can improve body composition and systemic metabolic parame-ters.11,12Resistance training may also promote favorable effects on cardiac function and exerci capacity,and this type of training is now recommended as a complementary exerci modality for patient with cardiovascular dia.13 Whereas it is widely appreciated that heart failure leads to muscle wasting,14a condition referred to as“cardiac ca-chexia,”it is unknown whether the rever is true;can an increa in lean muscle mass improve cardiac function in the tting of heart failure?
The Akt family of rine-threonine protein kinas are functionally redundant signaling molecules that are activated by various extracellular stimuli through the phosphatidylino-sitol3-kina pathway.Numerous studies have implicated Akt signaling in the control of organ size and cellular hypertrophy.15The Akt signaling pathway is preferentially activated in skeletal muscle in respon to anabolic stimuli and resistance training,9,16,17suggesting that Akt signaling may function as an important modulator of lean muscle mass through its ability to promote skeletal muscle hypertrophy and inhibit atrophy.18Consistent with the experimental obrvations,cardiac cachexia is associated with reduced Akt signaling in skeletal muscle tissue.19
In the prent study,we utilized a conditional transgenic (TG)mou model that can reversibly grow functional skeletal muscle by switching Akt1signaling specifically in skeletal muscle following permanent left anterior descending coronary artery(LAD)ligation to elucidate the role of fast/glycolytic skeletal muscle growth in the control of cardiac remodeling after the mou model of myocardial infarction(MI).We have previously reported that this model of Akt1overexpression in skeletal muscle leads to the lective growth of glycolytic,type IIb skeletal muscle hypertrophy.20This muscle is functional in that mice display an increa in grip strength,but spontaneous ambulatory activity and running performance of Akt1TG mice is lower than that of control mice.20Consistent with this phenotype, we have shown that transcripts for muscle-enriched glyco-lytic genes are significantly upregulated,whereas oxidative genes are reduced in Akt1TG mice.20Here,we demonstrate that Akt1–mediated,glycolytic skeletal muscle growth in-creas capillary growth in the myocardium and attenuates cardiac remodeling after MI.Skeletal muscle growth also incread in the cardiac eNOS signaling pathway,and inhi-bition of eNOS abolished the protective effects of muscle growth on post-MI cardiac remodeling.
Methods
Skeletal Muscle-Specific Conditional Akt1
TG Mice
The M-creatine kina(MCK)-rtTA TG mice21were crosd with TRE-myrAkt1TG mice22to generate double TG mice(Akt1TG mice).20Mice were maintained on mixed genetic backgrounds of C57BL/6J for MCK-rtTA TG and FVB and C57BL/6for TRE-myrAkt1TG mice and were bred to produce each t of Akt1TG experimental mice.The MCK promoter construct ud in the
driver line is mutated,and transgene expression is expresd in a
subt of muscle,but no expression occurs in heart.19,21For Akt1
transgene expression,Akt1TG mice were treated with doxycy-
cline(DOX)in drinking water,and DOX water was removed to
repress the transgene expression.MCK-rtTA single TG litter-
mates were ud as controls and treated with DOX in the same
manner as Akt1TG mice.Measurements of body composition
were made by quantitative magnetic resonance,as described
previously.23In some experiments,mice were given1mg/mL L-NAME(Dojindo)in drinking water at2days after MI or sham operation.Blood pressure of conscious mice was measured by
tail-cuff method(MK-2000ST,Muromachi,Japan)every2
weeks.Serum nitrogen oxide was measured by Nitrate/Nitrite
Colorimetric Assay Kit(Cayman Chemical Company). Mou Model of Myocardial Infarction
Control and Akt1TG mice at the ages of10to12weeks were
anesthetized with sodium pentobarbital(50mg/kg intraperitoneally).
The adequacy of anesthesia was monitored by the stability of blood
pressure and heart rate by tail-cuff method and the stability of
respiration.The trachea was cannulated with a polyethylene tube
connected to a respirator with a tidal volume of0.6mL(110/min).A
left thoracotomy was performed between the4th and5th ribs.The
pericardial tissue was removed,and LAD was visualized under a
microscope and permanently ligated with8-0silk sutures.24Sham-
treated mice underwent surgery but not LAD ligation.DOX treat-
ment started at2days after surgery.Survival of mice after MI was
evaluated using the Kaplan-Meier method.Survived mice were
euthanized after2or4weeks following DOX treatment.Mice were
anesthetized with overdo pentobarbital,and hearts and skeletal
muscle were rapidly excid and freeze-clamped for subquent
analys.All procedures were performed in accordance with the
Kumamoto University animal care guidelines(approval reference
No.B23-206),which conformed to the Guide for the Care and U
of Laboratory Animals,published by the US National Institutes of
Health(NIH Publication No.85-23,revid1996). Echocardiography
A transthoracic ultrasound cardiogram was performed using a Xario
system(Toshiba,Tokyo,Japan),equipped with a12-MHz linear
array transducer.M-mode images were recorded from the short-axis
view at the high papillary muscle level.Left ventricular end-diastolic
dimension(LVED),end-systolic dimension,intraventricular ptum,
往事随风飘and posterior wall thickness were measured.Fractional shortening
(%FS)was calculated using the following equation:%FSϭ(LVED-
丁丁历险记电影end–systolic dimension)/LVEDϫ100.All recordings were per-
formed in conscious mice.All echocardiography was performed by
investigators who were blinded to the identity of the mou genotype. Hemodynamic Measurements
Left ventricular contractile performance was determined4weeks after
DOX treatment,as described previously.25In brief,a1.4F catheter tip
micromanometer(Millar Instruments)was percutaneously introduced
into the carotid artery and gently advanced into the left ventricular cavity
in mice under pentobarbital anesthesia(25mg/kg single intraperitone-
ally injection).Pressure waves were recorded and analyzed with
LabChart7Pro software(AD Instruments).
Quantitative Real-Time PCR
Total RNA was prepared with a Qiagen RNeasy fibrous minikit,
using the protocol supplied by the manufacturer,and cDNA was
produced using ThermoScript RT-PCR Systems(Invitrogen,Carls-
bad,CA).Quantitative real-time PCR was performed,as described
previously.25Transcript expression levels were determined as the
number of transcripts relative to tho for36B4and normalized to
the mean value from control hearts.Table1lists the primer quences
ud in this study.
Araki et al Muscle Growth Attenuates Cardiac Remodeling117
买年货
Western Blot Analysis
Western blotting was performed with an SDS-PAGE Electrophoresis System,as described previously.25Primary antibody ud were:phospho-eNOS (Ser 1177,p-eNOS),total -eNOS (t-eNOS),and GAPDH from Cell Signaling Technology;VEGF-A from Abcam;␣-tubulin from Calbiochem.
Histological Analysis
Mice were euthanized and heart tissue was obtained at 2weeks after MI.Myocardial tissues were fixed in 4%paraformaldehyde,dehydrated and embedded in paraffin.To determine the infarct size,ctions were stained with Masson’s trichrome.Total LV circumference was calcu-lated as the sum of endocardial and epicardial gment lengths from all ctions.Infarct size was calculated as total infarct circumference divided by total LV circumference.Capillary density was assd by CD31staining of tissue ctions in the peri-infarct zone,as described previously.24Myocardial fibrosis and capillary density was quantified using Lumina Vision version 2.2analysis software.
Angiogenesis Antibody Array Analysis
The expression profile of 53angiogenesis-related proteins was analyzed with mou angiogenesis antibody arrays (R&D Systems Inc,Minne-apolis,MN).Blocking,hybridization of the array filters,washing conditions,and chemiluminescent detection steps were performed ac-cording to the instructions supplied by the manufacturer.
Statistical Analysis
All data are prented as mean ϮSEM.The differences among groups were evaluated by using linear mixed effects models with measurements values as respon variable,control/TG,and sham/MI,and the interactions as fixed effect and the parents as random effects.In multiple comparison tests,probability values were adjusted by Bonferroni method.Animal survival was evaluated by the Kaplan-Meier method,and the log-rank test was ud to compare survival curves
between
Figure 1.A ,Schematic illustration of experimental protocol and doxycycline (DOX)-treatment time cour.B ,Left ventricular (LV)diastolic dimension and percentage of fractional shortening in control and Akt1transgenic (TG)mice 2days after sham-operation or myocardial infarction (MI)(n ϭ7mice per experimental group).C ,Transgene expression following the addition of DOX.Repre-ntative blots of the gastrocnemius muscle and heart are shown.D ,Gastrocnemius muscle weight in control and Akt1TG mice at 2and 4weeks after DOX-treatment.E ,Left:Reprentative gross appearance of control and Akt1TG mice after 2and 4weeks of transgene induction.Right:Measurements of body composition after 2weeks of transgene induction were made by quantitative magnetic resonance.F ,Survival curves of control and Akt1TG mice after MI and sham.Control/sham,n ϭ15;Con-trol/MI,n ϭ20;Akt1TG/sham,n ϭ10;Akt1TG/MI,n ϭ10.Results are prented as mean ϮSEM.UCG indicates ultrasound cardio-gram;LAD,left anterior descending artery.
Table 1.Primer Sequences Ud for Quantitative Real-Time PCR
Gene Primer Sequences
BNP Forward 5Ј-GGAGTCCTAGCCAGTCTCC-3ЈRever 5Ј-TTGGTCCTTCAAGAGCTGTC-3Ј口红怎么画
Collagen I Forward 5Ј-GTCCCAACCCCCAAAGAC-3ЈRever 5Ј-CATCTTCTGAGTTTGGTGATACGT-3ЈCollagen III Forward 5Ј-GTCCCAACCCCCAAAGAC-3ЈRever 5Ј-CATCTTCTGAGTTTGGTGATACGT-3Ј36B4Forward 5Ј-GCTCCAAGCAGATGCAGCA-3ЈRever
5Ј-CCGGATGTGAGGCAGCAG-3Ј118Circ Heart Fail January 2012
偶数和奇数是什么意思MI-operated WT and MI-operated TG groups.Western blot densities were analyzed with Student t test.The significance level of a statistical hypothesis test was 0.05.
Results
Akt1–Mediated Skeletal Muscle Growth Attenuates Cardiac Dysfunction After MI
To investigate the relationships between skeletal muscle growth and cardiac remodeling,control (nontransgenic)and noninduced Akt1TG mice were subjected to sham surgery or permanent LAD ligation to induce MI (Figure 1A).At 2days following surgery and immediately before muscle-specific transgene in-duction with DOX,both control and Akt1TG mice exhibited a
progressive increa in LVED and a decrea in %FS relative to sham operated mice (Figure 1B).At
the time of the baline measurements,mice were provided with DOX in their drinking water.Mice were then harvested at either 2or 4weeks after DOX treatment to asss the progress of heart failure.
In this inducible transgenic system,Akt1transgene was detected in skeletal muscle but not in the heart,in respon to DOX treatment (Figure 1C).Transgene-induced skeletal muscle growth,as assd by analysis of the gastrocnemius muscle weight/body weight (BW)ratio,was incread at both 2and 4weeks after DOX treatment,and LAD ligation did not affect this parameter (Figure 1D).Whereas gastrocnemius muscle growth was substantial,the mutated MCK promoter ud in
the
Figure 2.A ,Top:Reprentative M-mode echocardiogram for control and Akt1transgenic (TG)mice 4weeks after sham-operation or myocardial infarction (MI).Bottom:Left ventricular (LV)diastolic dimension,LV systolic dimension,percentage of fractional shortening,intraventricular ptum,and posterior wall in control and Akt1TG mice 2and 4weeks after sham-operation or MI.B ,Systolic and dia-stolic arterial blood pressure and dP/dt max in control and Akt1TG mice 4weeks after sham-operation or MI.Results are prented as mean ϮSEM (n ϭ7mice per experimental group).
阅读感悟100字Table 2.Body Weight,Heart Rate,and Mean Arterial Pressure in Experimental Groups of Mice
Sham
MI Control
茶艺课程
Akt1TG P Value Control Akt1TG P Value BW (g)30.1Ϯ0.730.7Ϯ0.80.87629.5Ϯ0.730.3Ϯ1.10.343HR (bpm)670Ϯ11641Ϯ170.418687Ϯ12680Ϯ130.711mAP (mm Hg)
63.0Ϯ2.7
65.9Ϯ3.7
0.631
54.2Ϯ1.1
58.2Ϯ1.1
0.189
Results are prented as mean ϮSEM.Measurements were made at 4weeks post-surgery.
Araki et al Muscle Growth Attenuates Cardiac Remodeling 119
studies is expresd in a subt of myofibers(such as gastroc-nemius,tibialis anterior,and quadriceps muscle),and no trans-gene expression nor growth of other muscle groups including soleus and extensor digitorum extensor is obrved.21Thus,the overall extent of muscle growth in this model is modest,with an increa in lean mass of approximately5%as assd by QMR (Figure1E).
No significant difference occurred in the survival frequencies after MI between control and Akt1TG mice(Figure1F). Mortality in this model mostly occurred within10days of surgery,which was mainly ca
ud by cardiac rupture.Death from heart failure was rare in our experimental model,and only 1additional death was obrved until the termination of the experiment at4weeks after DOX treatment.BW and heart rate did not differ between control and Akt1TG mice at4weeks after DOX treatment in MI or sham treatment groups(Table2). Echocardiographic examination revealed that induction of the Akt1transgene for2or4weeks in skeletal muscle led to a decrea in LVED and end-systolic dimension(Figure2A).The protective effect of skeletal muscle Akt1expression on cardiac function and remodeling was more apparent at4weeks than at 2weeks after transgene activation,resulting in a statistically significant increa in%FS at the latter time point.DOX treatment of control mice had no effect on LV dimension and function in sham-operated mice at the time points. Systolic arterial pressure was significantly decread in both control and Akt1TG mice at4weeks after DOX treatment compared with sham-treated mice,but there was no statistical difference between the control and Akt1TG mice (Figure2B);however,left ventricular systolic function, reflected by dP/dt max,was improved in Akt1TG mice at4 weeks after DOX treatment(Figure2B).
Akt1–Mediated Skeletal Muscle Growth Prevents Cardiac Hypertrophy in Respon to MI
Cardiac tissue ctions were stained with Masson’s trichrome to calculate infarct size in control and DOX-treated,skeletal muscle-specific Akt1TG mice.The ratio of total infarct size to total LV circumfer
ence was the same in control and Akt1 TG mice.As shown in Figure3A,the increa in heart
Figure3.A,Heart weight/body weight
ratio in control and Akt1transgenic(TG)
mice at2and4weeks after doxycycline
(DOX)treatment.B,Left:Reprentative
images of hematoxylin and eosin-stained
heart ctions.Right:Quantitative analy-
sis of cardiomyocyte cross-ctional area
in control and Akt1TG mice2weeks
after sham-operation or myocardial infarc-
tion(MI).C,B-type natriuretic peptide
mRNA expression in control and Akt1TG
mice at2weeks after DOX treatment.D,
Lung wet weight/body weight ratio in
control and Akt1TG mice2and4weeks
after sham-operation or MI.Results are
prented as meanϮSEM(nϭ7mice per
experimental group).BZ indicates border
zone;RZ,remote zone.
120Circ Heart Fail January2012
weight/BW ratio at 2weeks after DOX treatment was significantly smaller in Akt1TG mice than that in control (5.0Ϯ0.1versus 5.5Ϯ0.1mg/g,P ϭ0.0332),and the differ-ence became more apparent at 4weeks after DOX treatment (4.8Ϯ0.4versus 5.8Ϯ0.2mg/g,P ϭ0.0088).No significant difference in hea
rt weight/BW ratio was obrved in sham-operated control versus Akt1TG mice.Analysis of the cardiomyocyte cross-ctional area in myocardial ctions of remote areas to the infarct revealed that cardiomyocyte hypertrophy in respon to MI was smaller in Akt1TG mice than in control mice (Figure 3B).MI led to an increa in the expression of the fetal-type cardiac gene BNP,and this upregulation was also attenuated in Akt1TG mice compared with control at 2weeks after DOX treatment (Figure 3C).Akt1activation in skeletal muscle did not detectably affect BNP expression in the hearts of sham-operated mice.Lung wet weight/BW ratio at 2and 4weeks after DOX treatment was significantly decread in Akt1TG mice compared with that in control mice,indicating diminished pulmonary con-gestion in mice expressing the skeletal muscle transgene (Figure 3D).
Akt1TG Mice Displayed Decread Interstitial Fibrosis and Incread Myocardial Capillary Density After MI
Myocardial interstitial fibrosis in the border and remote zone was assd in Masson S trichrome-stained tissue ctions (Figure 4A).Interstitial fibrosis was significantly decread in Akt1TG mice compared with control mice after MI.Little or no fibrosis could be detected in either Akt1TG or control mice that underwent sham surgeries.Consistent with the obrvations,collagen I and III gene expression in the border zone was significantly decread in the hearts of Akt1TG mice compared wi
th control mice (Figure 4A).Akt1activation in skeletal muscle did not
detectably affect collagen I and III expression in the hearts of sham-operated mice.
Capillary density was assd in histological ctions corre-sponding to the infarct border and remote zones (Figure 4C).CD31-positive cells in the heart incread following LAD ligation,and this increa was enhanced by Akt1transgene activation in skeletal muscle.Capillary density was unaffected by Akt transgene induction in sham-operated mice.
Akt1–Mediated Skeletal Muscle Growth Increas Angiogenesis Growth Factor Levels in Serum
Becau it is reported that myogenic Akt signaling controls angiogenic growth factor synthesis,26we examined the circulating angiogenic factors by the mou angiogenesis protein array and found that rum VEGF-A,FGF-1,FGF-2,and SDF-1levels were significantly upregulated in Akt1TG mice compared with control mice at 2weeks after DOX treatment (Figure 5A).Among them,the increa in rum VEGF-A concentration was validated by ELISA (87Ϯ7versus 69Ϯ11pg/mL,n ϭ6,P ϭ0.0229).VEGF-A protein expression was significantly incread in skeletal muscle of Akt1TG mice than that in control mice after 2or 4weeks DOX treatment (Figure 5B).There was no change in the cardiac expression of VEGF between control and Akt1TG hearts (data not shown).
Therapeutic Effects of Akt1–Mediated Muscle Growth on Cardiac Remodeling Is Blocked by the Treatment With NOS Inhibitor
Next,we assd whether Akt1–mediated skeletal muscle growth and subquent angiogenic factors’upregulation could affect intracellular signaling in the heart after MI.As shown in Figure 6A,the activating phosphorylation of eNOS at Ser1177,a downstream effector of angiogenic growth factor signaling,27was significantly incread in the border zone and remote
zone
Figure 4.A ,Left:Reprentative images of Masson’s trichrome-stained heart ctions.Right:Quantitative analysis of myocardial inter-stitial fibrosis in control and Akt1transgenic (TG)mice at 2weeks after transgene induction.B ,Collagen I,III mRNA expression in con-trol and Akt1TG mice 2weeks after sham operation or myocardial infarction (MI).C ,Top:Reprentative images of anti-CD31–stained heart ctions from remote zones.Bottom:Quantitative analysis of CD31–positive capillary density at border and remote zones in con-trol and Akt1TG mice at 2weeks after doxycycline (DOX)treatment.Similar gments of the hearts were analyzed in sham-operated mice.Results are prented as mean ϮSEM (n ϭ5mice per experimental group).
Araki et al Muscle Growth Attenuates Cardiac Remodeling 121
