Corsetti G v12 p362 Cell Met suppl 2010

Supplemental Information Cell Metabolism, Volume 12 Branched-Chain Amino Acid Supplementation Promotes Survival and Supports Cardiac and Skeletal Muscle Mitochondrial Biogenesis in Middle-Aged Mice Giuseppe D’Antona, Maurizio Ragni, Annalisa Cardile, Laura Tedesco, Marta Dossena, Flavia Bruttini, Francesca Caliaro, Giovanni Corsetti, Roberto Bottinelli, Michele O. Carruba, Alessandra Valerio, and

Enzo Nisoli

Figure S1, related to Figure 1. Plasma Branched-Chain Amino Acid Changes after BCAAem Bolus Administration

(A) Plasma of untreated WT (n = 4) and eNOS−/− (n = 4) mice was obtained before (t0) and at different time intervals (t1, 30 min; t2, 60 min; t3, 120 min) after a single bolus of BCAAem, correspoding to the daily supplementation dose (1.5 g/kg body weight) dissolved in tap water and administered by gavage. Data are expressed as mean ± SEM.

(B) Plasma concentrations of BCAAs are expressed as pmol/μl. Statistical significance was tested on Δ changes (t1-t0; t2-t0; t3-t0) by one-way ANOVA for repeated measures followed by Tukey post-hoc test. n.s., not statistically significant.

Pla

sma

conc

entra

tion

(pm

ol/µ

l)

leucineisoleucine valine

0500

100015002000

0250500750

1000

t0 t1 t2 t3

0500

1000150020002500 WT

eNOS-/-

t0 t1 t2 t3 t0 t1 t2 t3

A

BWT(t1-t0) -vs eNOS-/- (t1-t0) WT(t2-t0) -vs eNOS-/- (t2-t0) WT(t3-t0) -vs eNOS-/- (t3-t0)

mean diff 95% CI of diff P value mean diff 95% CI of diff P value mean diff 95% CI of diff P value

Isoleucine -8.420 -341.7 to 324.9 n.s. 61.0 -272.3 to 394.3 n.s. 113.5 -219.7 to 446.8 n.s.

Leucine -147.4 -816.1 to 521.3 n.s. 166.0 -502.7 to 834.7 n.s. 110.6 -558.1 to 779.4 n.s.

Valine -98.78 -758.8 to 565.2 n.s. 125.9 -536.1 to 787.9 n.s. -106.2 -768.2 to 555.8 n.s.

WT(t1-t0) -vs eNOS-/- (t1-t0) WT(t2-t0) -vs eNOS-/- (t2-t0) WT(t3-t0) -vs eNOS-/- (t3-t0)

mean diff 95% CI of diff P value mean diff 95% CI of diff P value mean diff 95% CI of diff P value

Isoleucine -8.420 -341.7 to 324.9 n.s. 61.0 -272.3 to 394.3 n.s. 113.5 -219.7 to 446.8 n.s.

Leucine -147.4 -816.1 to 521.3 n.s. 166.0 -502.7 to 834.7 n.s. 110.6 -558.1 to 779.4 n.s.

Valine -98.78 -758.8 to 565.2 n.s. 125.9 -536.1 to 787.9 n.s. -106.2 -768.2 to 555.8 n.s.

2

Figure S2, related to Figure 2. Various Amino Acid Mixtures Differentially Affect Mitochondrial Biogenesis in HL-1 Cardiomyocytes

(A, C, E, G, I) Mitochondrial DNA (mtDNA) content, analyzed by means of quantitative PCR and expressed as mtDNA copy number per nuclear DNA copy number, with value of untreated cells (open bars, Ctrl) taken as 1.0 (*p < 0.05, **p < 0.01, and ***p < 0.001 versus untreated cells; n = 5 experiments).

AArginine

0.0

3.0

mtD

NA

amou

nt(r

elat

ive

units

)2.0

1.0

0.1(mM)

1.0 10

BArginine

0.0

3.0

PG

C-1α

mR

NA

(rel

ativ

e ex

pres

sion

)

p = 0.0582.0

1.0

0.1(mM)

1.0 10

EMixture #1

0.0mtD

NA

amou

nt(r

elat

ive

units

) 2.0

1.0

0.01 0.1 1.0

F

0.0PGC

-1α

mR

NA

(rel

ativ

e ex

pres

sion

)2.0

1.0

0.01 0.1 1.0

GMixture #2

0.0mtD

NA

amou

nt(r

elat

ive

units

) 2.0

1.0

0.01 0.1 1.0

H

0.0PG

C-1α

mR

NA

(rel

ativ

e ex

pres

sion

)

2.0

1.0

0.01 0.1 1.0

I Mixture #3

0.0

3.0

mtD

NA

amou

nt(r

elat

ive

units

)

2.0

1.0

0.01 0.1 1.0

J Mixture #3

0.0

3.0

PG

C-1α

mR

NA

(rel

ativ

e ex

pres

sion

) ***

2.0

1.0

0.01 0.1 1.0

Mixture #1

Mixture #2

*******

* *

CCysteine

0.0mtD

NA

amou

nt(r

elat

ive

units

) 2.0

1.0

0.1 1.0 10

DCysteine

0.0PG

C-1α

mR

NA

(rel

ativ

e ex

pres

sion

)

2.0

1.0

0.1 1.0 10(mM)(mM)

3

(B, D, F, H, J) PGC-1α mRNA levels analysed by means of quantitative RT-PCR. The cycle number at which PGC-1α transcript was detectable was compared to that of 18S rRNA and expressed as relative values, with those measured in the untreated cells (open bars, Ctrl) taken as 1.0 (*p < 0.05, **p < 0.01, and ***p < 0.001 versus untreated cells; n = 5 experiments). All data represent mean ± SEM. Arginine and cysteine were used at 0.1–10 mM, while amino acid mixtures were used at 0.01X, 0.1X, or 1X (Table S1) final concentration.

4

Figure S3, related to Figure 3. BCAAem Supplementation Does Not Affect Mitochondrial Biogenesis in Adipose Tissues and Liver of WT Mice, Nor Does It Affect Mitochondrial Biogenesis and ROS Defense System in Different Tissues of eNOS−/− Mice

(A and B) PGC-1α, NRF-1, and Tfam (A) and SIRT1 (B) mRNA was analyzed by means of quantitative RT-PCR in white (WAT) and brown adipose tissue (BAT) of WT mice. Relative expression values measured in the untreated (open bars) sedentary mice were taken as 1.0 (n = 6 experiments; *p < 0.05 versus corresponding sedentary animals). S, sedentary mice; T. trained mice.

(C) Citrate synthase activity in WAT and BAT of WT mice. Values in untreated (open bars) sedentary (S) mice taken as 1.0 (n = 3 experiments; *p < 0.05 versus corresponding sedentary animals). T, trained mice.

(D) PGC-1α mRNA levels in liver of sedentary WT and eNOS−/− mice. Relative expression values measured in the untreated (open bars) WT mice were taken as 1.0 (n = 6 experiments; ***p < 0.001 versus untreated WT mice).

0

1

2m

RN

A(r

elat

ive

expr

essi

on)

AWAT

Sedentary TrainedBAT

0

1

2

Citr

ate

synt

hase

(vs.

unt

rete

dan

imal

s)

C

0

1

2

Mito

chon

dria

lDN

A(r

elat

ive

units

) E

Citr

ate

synt

hase

(vs.

unt

reat

edW

T m

ice)

***

0

F

***

PGC

-1α

mR

NA

(rel

ativ

e ex

pres

sion

)

0

1

2 WTD

eNOS-/-

***

1

2

0

1

2

mR

NA

(rel

ativ

e ex

pres

sion

)

PGC-1α

GSedentary

HEART

NRF-1Tfam

PGC-1αNRF-1

Tfam

Trained

PGC-1α

SedentaryDIAPHRAGM

NRF-1Tfam

PGC-1αNRF-1

Tfam

Trained

PGC-1α

SedentarySOLEUS

NRF-1Tfam

PGC-1αNRF-1

Tfam

Trained

PGC-1α

SedentaryTIBIALIS

NRF-1Tfam

PGC-1αNRF-1

Tfam

Trained

0

1

2

SIR

T1 m

RN

A(r

elat

ive

expr

essi

on)

HSedentary Trained

HEARTDIAPH

SOLEUS

TIBIALISHEART

DIAPH

SOLEUS

TIBIALIS

0

1

2

Citr

ate

synt

hase

vs.u

ntre

ated

anim

als

I

Sedentary Trained

HEARTDIAPH

SOLEUS

TIBIALISHEART

DIAPH

SOLEUS

TIBIALIS

Sedentary Trained

0

1

2

SIR

T1 m

RN

A(r

elat

ive

expr

essi

on)

BWAT BAT

S T S TWAT BAT

S T S T

PGC-1αNRF-1

Tfam

PGC-1αNRF-1

Tfam

PGC-1αNRF-1

Tfam

PGC-1αNRF-1

Tfam

* * * * *

WT eNOS-/- WT eNOS-/-

0

1

2

3

mR

NA

(rel

ativ

e ex

pres

sion

)

SOD1

HEART

SOD2

CatalaseGPx1

Sedentary

DIAPHRAGM

TrainedSedentary Trained

SOD1SOD2

CatalaseGPx1

SOD1SOD2

CatalaseGPx1

SOD1SOD2

CatalaseGPx1

SOD1SOD2

CatalaseGPx1

Sedentary TrainedSedentary Trained

SOD1SOD2

CatalaseGPx1

SOD1SOD2

CatalaseGPx1

SOD1SOD2

CatalaseGPx1

SOLEUS TIBIALISJ

5

(E) Mitochondrial DNA amount was analyzed by means of quantitative PCR in liver of sedentary WT and eNOS−/− mice. The relative units are expressed in comparison to those of untreated (open bars) WT mice taken as 1.0 (n = 5 experiments; ***p < 0.001 versus untreated WT mice).

(F) Citrate synthase activity in liver of sedentary WT and eNOS−/− mice. The values are expressed as fold-change versus untreated (open bars) WT mice taken as 1.0 (n = 3 experiments; ***p < 0.001 versus untreated WT mice).

(G and H) PGC-1α, NRF-1, and Tfam (G) and SIRT1 (H) mRNA levels in cardiac and skeletal muscles of eNOS−/− mice. Relative expression values measured in the untreated (open bars) sedentary mice were taken as 1.0 (n = 6 experiments).

(I) Citrate synthase activity in cardiac and skeletal muscles of eNOS−/− mice. Values in untreated (open bars) sedentary mice taken as 1.0 (n = 3 experiments.

(J) SOD1, SOD2, catalase, and GPx1 mRNA levels in cardiac and skeletal muscles of eNOS−/− mice. Relative expression values measured in the untreated mice (open bars) sedentary mice were taken as 1.0 (n = 6 experiments). All data represent mean ± SEM.

6

Figure S4, related to Figure 7. Involvement of eNOS and mTOR Pathways in BCAAem-Mediated Effects

(A-F) Mitochondrial biogenesis was analyzed in primary cardiac (A, C, and D) and gastrocnemius (B, E, and F) myocytes from WT and eNOS−/− mice. PGC-1α, NRF-1, Tfam, and copper/zinc superoxide dismutase (SOD1) mRNA (A and B) analyzed by means of quantitative RT-PCR. Relative expression values measured in the untreated cells (open bars) were taken as 1.0 (n = 5 experiments; *p < 0.05, **p < 0.01, and ***p < 0.001). Mitochondrial DNA amount was analyzed by means of quantitative PCR (C and E). The relative units are expressed in comparison to those of untreated cells (open bars) taken as 1.0 (n = 5 experiments; ***p < 0.001). Citrate synthase activity (D and F). Values of untreated cells (open bars) taken as 1.0 (n = 5 experiments; ***p < 0.001).

(G and H) Effect of rapamycin (20 nM) on BCAAem-mediated (closed bars) increase in mitochondrial biogenesis markers (G) and citrate synthase activity (H) in HL-1 cardiomyocytes (n = 3 experiments; *P < 0.05 and ***P < 0.001 versus corresponding BCAAem-untreated cells; †P < 0.05 versus BCAAem-treated cells without rapamycin).

0

1

2

Mito

chon

dria

lDN

A(r

elat

ive

units

)

WTC

Citr

ate

synt

hase

(vs.

unt

reat

edce

lls)

0

1

2

3

mR

NA

(rel

ativ

e ex

pres

sion

)

PGC-1α

A

*

WT

CARDIAC MYOCYTES

*

NRF-1Tfam

SOD 1

**

PGC-1αNRF-1

TfamSOD 1

eNOS-/-

*

0

1

2

3

mR

NA

(rel

ativ

e ex

pres

sion

)

PGC-1α

B

*

WT

GASTROCNEMIUS MYOCYTES

***

NRF-1Tfam

SOD 1

**

PGC-1αNRF-1

TfamSOD 1

eNOS-/-

**

eNOS-/-

3 ***

0

1

2

WTD eNOS-/-

3

***

0

1

2

Mito

chon

dria

lDN

A(r

elat

ive

units

)

WTE

Citr

ate

synt

hase

(vs.

unt

reat

edce

lls)

eNOS-/-

3

***

0

1

2

WTF eNOS-/-

3

***

0.0

4.0

mR

NA

(rel

ativ

e ex

pres

sion

)

3.0

2.0

***

G

1.0

PGC-1α NRF-1 Tfam

***

***

Rapamycin +- +- +-

* *

0.0

Citr

ate

synt

hase

(vs.

vehi

cle-

treat

edce

lls)

3.0

2.0***

H

1.0

Rapamycin +-

*

I Trained WT

Trained eNOS-/-

BCAAem - + - + - + - +p-mTOR

(Ser2448)

mTOR

HEARTDIAPH

SOLEUS

TIBIALIS

- + - + - + - +p-mTOR

(Ser2448)

mTOR

HEARTDIAPH

SOLEUS

TIBIALIS

BCAAem

7

(I) Effect of BCAAem supplementation on mTOR activation in middle-aged mice. BCAAem stimulated mTOR phosphorylation in cardiac muscle, diaphragm, and skeletal muscles of trained WT but not eNOS−/− mice. n = 3 experiments. All data represent mean ± SEM.

Figure S5, related to Figure 6. BCAAem Supplementation Does Not Reduce Oxidative Damage and Lipid Peroxidation in eNOS−/− Mice

(A and B) Mitochondrial H2O2 release, basal aconitase/total aconitase ratio, and superoxide dismutase activity (SOD) in heart (A) and soleus muscle (B) from eNOS−/− mice treated (closed bars) or not (open bars) with BCAAem (n = 10 experiments).

(C) Lipid peroxidation measured as malondialdehyde (MDA) production in skeletal muscle and white adipose tissue (WAT) from eNOS−/− mice. BCAAem-treated (closed bars) or not (open bars) animals (n = 5 experiments). All data represent mean ± SEM.

A

0.0

1.0

H2O

2(n

mol

/ min

/ mg

prot

ein)

0.5

0.0

1.5

Bas

alac

onita

se/

tota

l aco

nita

se

1.0

0

40

SOD

act

ivity

(U /

mg

prot

ein)

20

0.5

Heart

B

0.0

1.0

H2O

2(n

mol

/ min

/ mg

prot

ein)

0.5

0.0

1.5

Bas

alac

onita

se/

tota

l aco

nita

se

1.0

0

40

SO

D a

ctiv

ity(U

/ m

g pr

otei

n)

20

0.5

Soleus

C

MD

A (μ

mol

/ g)

50

100

250

200

150

0

100

400

300

200

0

MD

A (μ

mol

/ g)

MD

A (μ

mol

/ g)

10

30

60

50

40

0

20

Gastrocnemius Vastus WAT

8

Figure S6, related to Figure 5. Fiber Size of Skeletal Muscles, Endurance Capacity and Coordination Function in Untreated or BCAAem-Supplemented eNOS−/− Mice

(A) Fiber cross sectional area of skeletal muscles. V, vastus muscle; Gn, gastrocnemius muscle; Tib, tibialis muscle from eNOS−/− mice, untreated or supplemented with BCAAem (n = 5 mice per group). Grey bars, untreated adult mice; open bars, untreated middle-aged mice; black bars, BCAAem-treated middle-aged mice. †p < 0.001 versus adult mice; *p < 0.05 versus untreated middle-aged mice.

(B) Time to reach exhaustion following treadmill tests. BCAAem supplementation (closed bars) did not change endurance capacity of sedentary and trained eNOS−/− mice (n = 20 per group).

(C) Rotarod score. BCAAem supplementation (closed bars) did not significantly improve rotarod performance of sedentary and trained eNOS−/− mice (n = 10 experiments). All data represent mean ± SEM.

A

Fibe

r cro

ss s

ectio

nal a

rea

(μm

2 )

0

3000

1000

V Gn Tib

*2000 *

B C

0

30

20

10

Sedentary Trained

Tim

e to

exha

ustio

n(m

in)

0

300

200

100

Sedentary Trained

Sco

re (s

)

9

Table S1. Detailed Composition of Amino Acid Mixtures

Mixture #1 Mixture #2 Mixture #3

% C % C % C

L-Alanine 2.0 8.9 9.5 42.0

L-Arginine 6.6 29.4 4.6 20.5

L-Asparagine 3.5 15.8 2.1 9.3

L-Aspartic acid 2.0 8.9 1.1 4.7

L-Cysteine 2.0 8.9 1.9 8.6 3.8 16.7

L-Glutamic acid 16.0 70.9 3.9 17.3

L-Glutamine 13.6 60.3

Glycine 13.8 61.2 6.0 26.5

L-Histidine 2.0 8.7 2.5 11.0 3.8 16.7

L-Isoleucine 4.9 21.5 3.5 15.5 15.6 69.4

L-Leucine 6.6 29.2 8.7 38.6 31.3 138.8

L-Lysine 8.5 37.8 11.6 51.7 16.2 72.1

L-Methionine 8.5 37.6 1.6 7.0 1.3 5.6

L-Phenylalanine 6.9 30.5 2.2 9.7 2.5 11.1

L-Proline 2.0 8.9 3.1 13.6

L-Serine 2.0 8.9 5.6 24.8

L-Threonine 4.9 21.5 5.7 25.3 8.8 38.9

L-Tryptophan 1.1 4.7 3.8 16.8 0.5 2.2

L-Tyrosine 2.0 8.9 3.6 16.0 0.7 3.3

L-Valine 4.9 21.5 5.6 24.8 15.6 69.4

Compositions are expressed either as percentage of total amino acid amount (%, g/100 g) or

as final 1X concentration (C, mg/100 ml) in cultured cells.

10

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Diet Amino Acid Content

Amino acid content of the standard diet (Laboratorio Dottori Piccioni, Gessate,

Italy) are reported as g/100 g mice food: arginine, 1.15; histidine, 0.48; isoleucine,

0.80; leucine, 1.50; lysine, 1.05; methionine + cysteine, 0.75; phenylalanine, 0.90;

threonine, 0.75; tryptophan, 0.23; valine, 1.00. Methionine was supplemented to the

basic mixture.

Plasma Hormone and Glucose Levels

Blood samples were collected between 10:00 a.m. and 12:00 a.m. under nonfasting

conditions in aged mice. Insulin concentrations were measured with an ELISA kit

(Linco Research Inc) (sensitivity, 0.2 ng/ml; intra-assay variation, 0.92%; interassay

variation, 6.03%); IGF-1 and GH concentrations were measured with ELISA kits

(Diagnostic Systems Laboratories) according to the manufacturer’s instructions

(IGF-1, sensitivity 1.3 ng/ml; intra-assay variation, 4.83%; interassay variation

7.0%; GH, sensitivity 1 ng/ml; interassay variation, 7.0%). Total testosterone levels

were determined by solid-phase immunoassay using a kit (Diagnostic Products

Corporation; Los Angeles, CA) and DPC Immulite 2000 immunoassay analyzer

(detection limit, 0.5 nmol/l; interassay variation < 7%; cross-reactivity with cortisol

and estradiol, 0.005% and 0.02%, respectively).

Plasma BCAA Levels

To study the BCAA absorption rate, plasma was obtained from WT and eNOS−/−

mice before (t0) and at different time intervals (t1, 30 min; t2, 60 min; t3, 120 min)

after a single bolus of BCAAem, correspoding to the daily supplementation dose

(1.5 g/kg body weight) dissolved in tap water and administered by gavage. BCAA

analysis was performed as previously described (Aquilani et al., 2000). Plasma was

mixed with internal standards, dried in glass tubes in a vacuum concentrator and

subjected to vapor phase hydrolysis by 6N HC1 at 110°C for 24 hr under argon

atmosphere in the presence of phenol. The samples were subsequently reconstituted

in pH 10.4 borate buffer and transferred to a Hewlett Packard AminoQuant II system

11

for automated derivatization and loading. The AminoQuant analyzed peptides and

proteins by precolumn derivatization of hydrolyzed samples with o-phthalaldehyde

(OPA) and 9-fluoromethyl-chloroformate (FMOC). Derivatized BCAAs were

separated by reverse-phase HPLC and detected by UV absorbance with a diode

array detector or by fluorescence using an in-line fluorescence detector at 338

excitation and 450 nm emission for OPA and 262 nm excitation and 305 nm

emission for FMOC. Borate buffer, OPA and FMOC reagents, amino acid standards,

200 x 2.1 mm C-18 reverse phase columns (5 mm particle size) and precolumns

were purchased from Hewlett-Packard. The BCAA plasma concentration were

expressed as pmol/μl.

HL-1 Cell Cultures and Treatments

HL-1 cardiomyocytes (a gift from W.C. Claycomb) (Claycomb et al., 1998) were

plated in fibronectin/gelatin-coated flasks, grown to 70%–80% confluence in

Claycomb medium (JRH Biosciences) supplemented with 100 μM norepinephrine

(from a 10 mM norepinephrine [Sigma-Aldrich] stock solution dissolved in 30 mM

L-ascorbic acid [Sigma-Aldrich]), 2 mM L-glutamine, 100 U/ml penicillin, 100

μg/ml streptomycin and 10% FBS (JRH Biosciences) (Claycomb et al., 1998). HL-1

cardiomyocytes were treated with single amino acids or different amino acid

mixtures (amino acids were individually obtained from Sigma-Aldrich) for 48 hr,

except for time-response experiments. In particular, we analyzed the in vitro effects

of L-arginine and L-cysteine, which were described to increase mitochondrial

biogenesis in mammalian cells (Fu et al., 2005) and to ameliorate insulin sensitivity

in rats (Blouet et al., 2007), respectively. Moreover, the effects of previously

described amino acids mixtures were analyzed (Table S1): a 40% restricted dietary

amino acids (except methionine) mixture (mixture #1), which was showed to

decrease mitochondrial protein oxidative modification, and increase SIRT1 in rat

liver (Caro et al., 2009); the mixture used by Patti et al. (1998) (mixture #2) in

which the final concentrations of amino acids were approximately equal to, or

multiples of, the normal plasma concentrations found in portal vein of starved rats;

and the BCAA-enriched mixture (BCAAem, mixture #3) which was suggested to

improve muscle function and glucose metabolism in aged humans and rodents

(Pansarasa et al., 2008; Solerte et al., 2008b). Medium osmolarity was measured

12

with a OM-6050 Osmo Station (Menarini). Osmolarity of complete medium was

304 ± 3 mOsm/kg; medium with 0.1X BCAAem, 305 ± 2 mOsm/kg; medium with

1X BCAAem, 349 ± 4 mOsm/kg. Exposure of HL-1 cells to sucrose to produce a

final medium osmolarity of 350 ± 3 mOsm/kg did not affect mtDNA content nor

expression of mitochondrial biogenesis markers. For the study of phospho-proteins,

HL-1 cells were serum-starved for 4 hr, then treated with BCAAem in serum-free

medium for 30 min unless otherwise specified. To investigate the intracellular

pathways involved in amino acid effects on mitochondrial biogenesis, HL-1 cells

were treated with vehicle (0.002% DMSO) or rapamycin (starting 30 min before the

addition of amino acids until the end of incubation time).

Endothelial NOS Gene Silencing

For eNOS knockdown experiments, HL-1 cells were transfected with 30 nM eNOS

siRNA SMARTpool (Dharmacon) or siGENOME nontargeting siRNA using

Dharmafect 3 transfection reagent. Efficacy of transfection was determined using

siGLO-RISC-free nontargeting siRNA and estimation of siRNA uptake by

fluorescence detection (absorbance/emission 557/570 nm). At the end of the

treatments, HL-1 cells were harvested as described below for further analysis.

Cardiac Myocyte Isolation, Culture, and Treatment

Adult cardiac myocytes were isolated from the left ventricle (LV) free wall of both

WT and eNOS−/− mice (Zhou et al., 2000). Briefly, mice were heparinized (1,500

U/kg i.p.) and anesthetized (pentobarbital sodium, 100 mg/kg i.p.). The heart was

excised, mounted on a steel cannula, and retrograde perfused at constant pressure

(100 cm H2O) at 37°C for ~3 min with a Ca2+-free bicarbonate-based buffer

containing 120 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4,

5.6 mM glucose, 20 mM NaHCO3, 10 mM 2,3-butanedione monoxime (Sigma), and

5 mM taurine (Sigma), previosuly gassed with 95% O2-5% CO2. Enzymatic

digestion was performed by adding collagenases B and D, protease type XIV. After

~3 min of digestion, 50 µM Ca2+ was added to the enzyme solution. About 7 min

later, the LV was quickly removed, cut into several chunks, and further digested in a

shaker (60-70 rpm) for 10 min at 37°C in the same enzyme solution. The supernatant

containing the dispersed myocytes was filtered into a sterilized tube and centrifuged

13

at 500 rpm for 1 min. The pellet was then resuspended in buffers containing

progressively increased concentration of Ca2+ from 0.05 to 0.125 to 0.25 to 0.5 mM

in 3 steps (10 min interval each). Freshly isolated cardiac myocytes were washed 3

times with minimal essential medium Eagle (MEM, Sigma M1018) containing 1.2

mM Ca2+, 2.5% FBS (Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin.

Then myocytes were plated at 0.5–1 x 104 cells/cm2 in culture dishes precoated with

10 μg/ml mouse laminin with MEM containing 2.5% FBS and antibiotics. After 1 hr

of culture in a 5% CO2 incubator at 37°C, the medium was changed to FBS-free

MEM. The day after seeding, cardiac myocytes were exposed to BCAAem for 48 hr

as described for HL-1 cells.

Skeletal Myocyte Culture and Treatment

Mouse satellite cells were prepared from 1-2 week postnatal mouse limbs of WT

and eNOS−/− mice as previously described (Cossu et al., 1980). The cells were

grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 20%

horse serum (HS, Invitrogen) and 5% chicken embryo extract (EE, Seralab). To

induce differentiation, the cells were shifted to DMEM supplemented with 5% HS

and 1.25% EE for 3-5 days, then cells were exposed to this culture medium

containing the BCAAem (as for HL-1 cells) for 48 hr.

Quantitative RT-PCR

Total RNA was extracted from cells or tissues using the RNeasy Lipid (or Fibrous)

Tissue Mini Kit (QIAGEN) and subjected to on-column DNAse digestion according

to manufacturer’s instructions. One microgram of total RNA was reverse transcribed

using iScript cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR reactions were

performed as described (Tedesco et al., 2008) and run with the iQ SybrGreenI

SuperMix (Bio-Rad) on an iCycler iQ Real-Time PCR detection system (Bio-Rad).

Calculations were performed by a comparative method (2-ΔΔCt) using 18S rRNA as

an internal control. Primers (sequences reported in table below) were designed using

Beacon Designer 2.6 software (Premier Biosoft International).

14

Immunoblot Analysis

Protein extracts were obtained by harvesting cultured cells in M-PER Mammalian

Protein Extraction Reagent or homogenizing tissues in T-PER Tissue Protein

Extraction Reagent (both from Pierce Biotechnology) as indicated by the

manufacturer, in the presence of 1 mM NaVO4, 10 mM NaF and a cocktail of

protease inhibitors (Sigma-Aldrich). Western blotting was performed as described

(Tedesco et al., 2008). Primary antibodies were: anti-SIRT1 (1:200, Upstate

Biotechnology); anti-GAPDH (1:10,000, Histoline Laboratories); anti phospho-

eNOS (Ser1177) and anti-eNOS (both 1:500 dilution, Transduction Labs); anti-

phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-70-S6 kinase (Thr389), anti-

70-S6 kinase, anti-phospho-4E-BP1 (Thr37/46), anti-4E-BP1 (all 1:1,000 dilution,

Cell Signaling Technology). After the visualization of phospho-proteins, filters were

stripped with the Re-Blot Western blot recycling kit (Chemicon International,

Tamecula, CA) and further used for the visualization of total (phosphorylated and

not phosphorylated) proteins.

Mitochondrial DNA Analysis

Mitochondrial DNA amount was measured by means of quantitative PCR as

previously described (Tedesco et al., 2008). Total DNA was extracted with QIAamp

DNA extraction kit (QIAGEN). Mitochondrial DNA was amplified using primers

specific for the mitochondrial cytochrome B (CytB) gene and normalized to

genomic DNA by amplification of the large ribosomal protein p0 (36B4) nuclear

gene.

ATP Measurement

The whole amount of ATP in cells was measured by using the ATP determination

kit from Molecular Probes as described (Tedesco et al., 2008). Cells were harvested

by trypsinization and centrifuged at 500 g for 5 min at 4°C. ATP was extracted by

incubating cell pellets with 1% trichloroacetic acid/4 mM EDTA solution for 10 min

on ice. Cell extracts were centrifuged at 12,000 g for 10 min at 4°C and used for

ATP determination as indicated by the manufacturer.

15

Citrate Synthase Activity

The activity was measured spectrophotometrically at 412 nm at 30°C in either

tisssue or whole cell extracts (Tedesco et al., 2008). Tissue or cell homogenates

were added to buffer containing 0.1 mM 5,5-dithio-bis-2-nitrobenzoic acid, 0.5 mM

oxaloacetate, 50 μM EDTA, 0.31 mM acetyl CoA, 5 mM triethanolamine

hydrochloride, and 0.1 M Tris-HCl, pH 8.1. Citrate synthase activity was measured

as nmol citrate produced/min/mg protein.

Electron Microscopy

Heart and soleus muscle were carefully removed, cut into pieces of about 1 mm3,

and placed in ice-cold fixative (2% glutaraldehyde in 0.1 M sodium-cacodylate

buffer, pH 7.4) for 5 hr. Samples were then extensively washed with 0.1 M

cacodylate buffer, postfixed for 2 hr with 2% OsO4, 0.1 M cacodylate buffer,

dehydrated in ethanol, block-stained with uranyl acetate, and embedded in Epon.

Ultrathin sections, collected on copper grids, were doubly stained with uranyl

acetate and lead citrate and examined under a Philips CM10 transmission electron

microscope. Morphometric analysis of mitochondria was performed as described

(Weibel, 1979). Randomly selected areas of tissue derived from three animals per

group were photographed at a magnification of 5,200 x and analyzed with NIH

Image Software. Statistical analyses of cross-sectional area of mitochondria and

mitochondrial densities were carried out using a Prism 2.0 software.

Mitochondrial Aconitase and SOD Activity Assays

Mitochondria were isolated using the Qproteome Mitochondria isolation kit

(QIAGEN). To measure aconitase specific activity, the formation of NADPH was

followed spectrophotometrically (340 nm) at 25°C as previously described (Lionetti

et al., 2007). SOD activity was measured with the Superoxide Dismutase Assay Kit

(Calbiochem). One unit of SOD activity is defined as the amount of enzyme needed

to exhibit 50% dismutation of the superoxide radical.

Mitochondrial H2O2 Production

Mitochondria were isolated as above. Mitochondrial H2O2 release was measured in

the presence of horseradish peroxidase using the Amplex Red Hydrogen

16

Peroxide/Peroxidase Assay kit (Molecular Probes). Fluorimetric measures were

made using a Fusion Universal Microplate Analyzer (Packard) with excitation filter

at 550 nm and emission filter at 590 nm. H2O2 production was calculated from a

standard curve generated from known concentrations of H2O2.

Lipid Peroxidation Assay

Lipid peroxidation (LPO) was used as an indicator of oxidative stress in muscle

tissues. Measurement of malondialdehyde (MDA) derived from polyunsaturated

fatty acid peroxides was evaluated spectrophotometrically at 586 nm by means of

the LPO-586 colorimetric assay kit (OxisResearch). LPO levels were expressed as

μM MDA/mg protein.

Training, Exercise Tolerance, and Motor Coordination Tests

Exercise training and exercise exhaustion tests were performed on the belt of a 6

lanes motorized treadmill (Exer 3/6 Treadmill, Columbus Instruments), supplied

with shocker plates (electrical stimulus: 200 ms, 0.34 mA, 1 Hz). For exercise

training, after one daily acclimation session of 20 min at 6 m/min for 2 days,

animals ran 5 days/week for 4 weeks (first week, 30 min at 10 m/min; second week,

60 min at 10 m/min; third and fourth week, 60 min at 12 m/min). The day after the

last session of exercise training, mice were sacrified. The last week before sacrifice

mice were subjected to exhaustion treadmill tests at 0° inclination as previously

described (Benchaouir et al., 2007).

Constant speed rotarod (47600 Model, Ugo Basile) was used to measure fore- and

hind-limb motor coordination and balance. Sedentary and trained, BCAAem-

supplemented or not, mice were studied at 12 months of age, receiving three trials

per day for two consecutive days. The mice were placed on the rod and trained at an

initial constant speed of 8 rpm for 5 min; then the speed was increased to 30 rpm for

5 min and the latency to fall was recorded. Time score was measured as the mean of

the individual best performances over the three trials at the second day (Serradj and

Jamon, 2007).

17

Table S2. Primers Used for PCR Analysis

Gene Primer Sequence Ta (°C) PCR product (bp)

PGC-1α Sense 5’-ACTATGAATCAAGCCACTACAGAC-3’

61 148 Antisense 5’-TTCATCCCTCTTGAGCCTTTCG-3’

Nrf-1

Sense 5’-ACAGATAGTCCTGTCTGGGGAAA-3’ 61 99 Antisense 5’-TGGTACATGCTCACAGGGATCT-3’

Tfam

Sense 5’-AAGACCTCGTTCAGCATATAACATT-3’ 61 104 Antisense 5’-TTTTCCAAGCCTCATTTACAAGC-3’

β-F1-ATPase Sense 5’-CGTGAGGGCAATGATTTATACCAT-3’

62 170 Antisense 5’-TCCTGGTCTCTGAAGTATTCAGCAA -3’

Cyt c Sense 5’-ATAGGGGCATGTCACCTCAAAC-3’

60 172 Antisense 5’-GTGGTTAGCCATGACCTGAAAG-3’

Cox IV Sense 5’-GTGGTTAGCCATGACCTGAAAG-3’

60 113 Antisense 5’-TTAGCATGGACCATTGGATACGG-3’

SIRT1 Sense 5’-ACGGTATCTATGCTCGCCTTG-3’

60 150 Antisense 5’-GACACAGAGACGGCTGGAAC-3’

SOD1 Sense 5’-GGCTTCTCGTCTTGCTCTC-3’

60 153 Antisense 5’-AACTGGTTCACCGCTTGC-3’

SOD2 Sense 5’-GCCTCCCAGACCTGCCTTAC-3’

63 131 Antisense 5’-GTGGTACTTCTCCTCGGTGGCG-3’

Catalase Sense 5’-CACTGACGAGATGGCACACTTTG-3’

63 173 Antisense 5’-TGGAGAACCGAACGGCAATAGG-3’

GPx1 Sense 5’-TCTGGGACCTCGTGGACTG-3’

62 157 Antisense 5’-CACTTCGCACTTCTCAAACAATG-3’

18S

Sense 5’-CTGCCCTATCAACTTTCGATGGTAG-3’ 60 100 Antisense 5’-CCGTTTCTCAGGCTCCCTCTC-3’

Cyt B Sense 5’-CTTCGCTTTCCACTTCATCTTACC-3’

61 92 Antisense 5’-TTGGGTTGTTTGATCCTGTTTCG-3’

36B4

Sense 5’-AGGATATGGGATTCGGTCTCTTC-3’ 61 143 Antisense 5’-TCATCCTGCTTAAGTGAACAAACT-3’

Ta, temperature of annealing.

18

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Cossu, G., Zani, B., Coletta, M., Bouche, M., Pacifici, M., and Molinaro, M. (1980). In vitro differentiation of satellite cells isolated from normal and dystrophic mammalian muscles. A comparison with embryonic myogenic cells. Cell Differ. 9, 357-368.

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