Ventricular Function Myocardial Hypertrophy and Improves ... · Activation of Serine One-Carbon...

Activation of Serine One-Carbon

Metabolism b y Calcineurin Ab 1 Reduces

Myocardial Hypertrophy and Impro ves

Ventricular Function

Laura Padrón-Barthe, P D,Ha b c, , María Villalba-Orero, P D,H

a c, Jesús M. Gómez-Salinero, P D,Ha Rebeca Acín-Pérez, P D,H

a

Sara Cogliati, P D,Ha Marina López-Olañeta, MLT, a Paula Ortiz-Sánchez, BS ,C

a Elena Bonzón-Kulichenko, P D,Ha c,

Jesús Vázquez, P D,Ha b, Pablo García-Pavía, MD, P D,H

b c d, , Nadia Rosenthal, P D,He f, José Antonio Enríquez, P D,H

a

Enrique Lara-Pezzi, P DHa c f, ,

A B S T R A C T

BACKGROUND In response to pressure overload, the heart develops ventricular hypertrophy that progressively de-

compensates and leads to heart failure. This pathological hypertrophy is mediated, among others, by the phosphatase

calcineurin and is characterized by metabolic changes that impair energy produ ction by mitochondria.

OBJECTIVES The authors aimed to determine the role of the calcineurin splicing variant CnAb 1 in th e context of cardiac

hypertrophy and its mechanism of action.

METHODS Transgenic mice overexpressing CnAb 1 speci cally in cardiomyocytes and mice lacking the unique C-terminalfi

domain in CnAb1 ( CnAb1Di12 mice) were used. Pressure overload hypertrophy was induced by transaortic constriction.

Cardiac function was measured by echocardiograph y. Mice were characterized using various molecu lar analyses.

RESULTS In contrast to other calcineurin isoforms, the authors show here that cardiac-speci c overexpression of CnAfi b1

in transgenic mice reduces cardiac hypertrophy and improves cardiac function. This effect is mediated by activation of

serine and one-carbon metabolism, and the production of antioxidant mediators that prevent mitochondrial protein

oxidation and preserve ATP production. The induction of enzymes involved in this metabolic pathway by CnAb1 i s

dependent on mTOR activity. Inhibition of serine and one-carbon metabolism blocks the bene cial effects of CnAfi b1.

CnAb1Di12 mice show increased cardiac hypertrophy and declined contractili ty.

CONCLUSIONS The metabolic reprogramming induced by CnAb 1 rede nes the role of calcineurin in thefi

heart and shows for the rst time that activation of the serine and one-carbon pathway has bene cial effects onfi fi

cardiac hypertrophy and function, paving the way for new therape utic approaches. (J Am Coll Cardiol 2018;71:654 67)–

© 2018 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open

access article under the CC BY-NC-ND license ( ).http://creativecommo ns.org/licenses/by-nc-nd/4.0/

ISSN 0735-1097 https://doi.org/10.1016/j.jacc.2017.11.067

From the a Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain; b Heart Failure and Inherited Cardiac

Diseases Unit, Department of Cardiology, Hospital Universitario Puerta de Hierro, Madrid, Spain; c Centro de Investigacion Bio-

medica en Red Cardiovascular (CIBERCV), Madrid, Spain; d Facultad de Ciencias de la Salud, Universidad Francisco de Vitoria,

UFV, Pozuelo de Alarcón, Madrid, Spain; e The Jackson Laboratory, Bar Harbor, Maine; and the f National Heart and Lung Institute,

Imperial College London, United Kingdom. This work was supported by grants from the European Union (CardioNeT-ITN-289600

and CardioNext-608027 to Dr. Lara-Pezzi; Meet-ITN-317433 to Dr. Enríquez; UE0/MCA1108 to Dr. Acín-Pérez), from the Spanish

Ministry of Economy and Competitiveness (SAF2015-65722-R and SAF2012-31451 to Dr. Lara-Pezzi; SAF2015-71521-REDC, BFU2013-

50448, and SAF2012-32776 to Dr. Enríquez; RyC-2011-07826 to Dr. Acín-Pérez; BIO2012-37926 and BIO2015-67580-P to Dr. Váz-

quez), from the Spanish Carlos III Institute of Health (CPII14/00027 to Dr. Lara-Pezzi; RD12/0042/066 to Drs. García-Pavía and

Lara-Pezzi), from the Regional Government of Madrid (2010-BMD-2321 Fibroteam to Dr. Lara-Pezzi; 2011-BMD-2402 Mitolab to“ ” “ ”

Dr. Enríquez) and the FIS-ISCIII (PRB2-IPT13/0001 and RD12/0042/0056-RIC-RETICS to Dr. Vázquez). This work was also sup-

ported by the Plan Estatal de I D I 2013-2016 European Regional Development Fund (FEDER) A way of making Europe,þ þ – “ ”

Spain. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and by the Pro-CNIC Foundation and is a

Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). Drs. Váz quez and García -Pavía have served as consultants for VL39.

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J O U R N A L O F T H E A M E R I C A N C O L L E G E O F C A R D I O L O G Y V O L . 7 1 , N O . 6 , 2 0 1 8

ª 2 0 1 8 T H E A U T H O R S . P U B L I S H E D B Y E L S E V I E R O N B E H A L F O F T H E A M E R I C A N

C O L L E G E O F C A R D I O L O G Y F O U N D A T I O N . T H I S I S A N O P E N A C C E S S A R T I C L E U N D E R

T H E C C B Y - N C - N D L I C E N S E ( h t t p : / / c r e a t i v e c o m m o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / ) .

C a r d i a c h y p e r t r o p h y p r o g r e s s i v e l y d e c o m -

p e n s a t e s a n d b e c o m e s m a l a d a p t i v e , l e a d i n g

t o p a t h o l o g i c a l c a r d i a c r e m o d e l i n g a n d h e a r t

f a i l u r e ( 1 ) . M a l a d a p t i v e c a r d i a c h y p e r t r o p h y i s a c c o m -

p a n i e d b y i n t e r s t i t i a l a n d p e r i v a s c u l a r fi b r o s i s a n d b y

c h a n g e s i n c a r d i a c m e t a b o l i s m . T h e h y p e r t r o p h i c

h e a r t p r o g r e s s i v e l y r e v e r t s t o a n e m b r y o n i c m e t a b o l i c

p r o g r a m w i t h r e d u c e d f a t t y a c i d o x i d a t i o n a n d

i n c r e a s e d r e l i a n c e o n g l u c o s e m e t a b o l i s m t h a t r e s u l t

i n d e c r e a s e d A T P p r o d u c t i o n ( 2 ) .

T h e c a l c i u m - r e g u l a t e d p h o s p h a t a s e c a l c i n e u r i n

p l a y s a m a j o r r o l e i n p a t h o l o g i c a l h y p e r t r o p h y . C a l -

c i n e u r i n i s c o m p o s e d o f a c a t a l y t i c ( C n A ) a n d a r e g -

u l a t o r y ( C n B ) s u b u n i t . C n A i s e n c o d e d b y 3 d i f f e r e n t

g e n e s ( r e s u l t i n g i n C n Aa, C n Ab , a n d C nAg ) , w i t h

C n Ab b e i n g t h e m a i n i s o f o r m i n t h e h e a r t . T w o s p l i c e

v a r i a n t s f o r C n Ab h a v e b e e n d e s c r i b e d . A l t h o u g h

C n Ab 2 h a s a C - t e r m i n a l a u t o i n h i b i t o r y d o m a i n a n d

a c t s l i k e a t y p i c a l C n A , C n Ab 1 h a s a u n i q u e C - t e r m i n a l

d o m a i n , n o t s h a r e d b y a n y o t h e r k n o w n p r o t e i n , t h a t

c o n f e r s t h e s e i s o f o r m s p e c i fi c p r o p e r t i e s ( 3 – 6 ) .

C o n s t i t u t i v e a c t i v a t i o n o f c a l c i n e u r i n o r i t s m a i n

t a r g e t , t h e t r a n s c r i p t i o n f a c t o r n u c l e a r f a c t o r o f a c t i -

v a t e d T c e l l s ( N F A T c ) , l e a d s t o m a s s i v e m a l a d a p t i v e

c a r d i a c h y p e r t r o p h y ( 7 ) . B y c o n t r a s t , m i c e l a c k i n g

C n Ab s h o w r e d u c e d v e n t r i c u l a r h y p e r t r o p h y i n

r e s p o n s e t o p r e s s u r e o v e r l o a d ( 8 ). H o w e ve r , t h e r o l e

o f C n Ab 1 i n t h i s c o n t e x t i s u n k n o w n .

M E T H O D S

F u l l M e t h o d s c a n b e f o u n d i n t h e O n l i n e A p p e n d i x .

M I C E . aM H C - C n Ab 1 m i c e e x p r e s s t h e h u m a n C n Ab1

i s o f o r m i n a c a r d i o m y o c y t e - r e s t r i c t e d m a n n e r u n d e r

t h e c o n t r o l o f t h e a l p h a m y o s i n h e a v y c h a i n (aM H C )

p r o m o t e r ( 4 ) . C n Ab1Di 1 2 m i c e w e r e g e n e r a t e d b y d e -

l e t i n g i n t r o n 1 2 - 1 3 i n t h e g e n e t h a t e n c o d e s C n Ab

(P p p 3 c b ) , w h i c h e n c o d e s t h e u n i q u e C - t e r m i n a l

d o m a i n i n C n Ab1 . O n l y a d u l t m a l e m ic e we r e u s e d i n

t h i s s t u d y . A l l p r o c e d u r e s w e r e a p p r o v e d b y t h e

e t h i c s c o m m i t t e e s o f t h e C N I C a n d t h e R e g i o n a l

G o v e r n m e n t o f M a d r i d .

S U R G E R I E S A N D E C H O C A R D I O G R A P H I C A N A L Y S I S .

M a l a d a p t i v e c a r d i a c h y p e r t r o p h y w a s i n d u c e d b y

t r a n s a o r t i c c o n s t r i c t i o n ( T A C ) t r y i n g t o r e p r o d u c e t h e

h u m a n c o n d i t i o n a s m u c h a s p o s s i b l e ( 9 ) . W h er e

i n d i c a t ed , L - b u t h i o ni n e - s u l f o x i m i n e ( B S O )

( 3 g / l i n d r i n k i n g w a t e r ) o r N C T - 5 0 3

( 0 . 9 m g / m o u s e , d a i l y i n t r a p e r i t o n e a l i n j e c -

t i o n ) w a s a d m i n i s t e r e d f o r 2 1 d a y s , s t a r t i n g

o n t h e d a y o f t h e s u r g e r y . T r a n s t h o r a c i c

e c h o c a r d i o gr a p h y w a s p e r f o r m e d b l i n d l y u s -

i n g a n u l t r a – h i g h - r e s o l u t i o n e c h o c a r d i o g r a -

p h y s y s t e m w i t h a l i n e a r 3 0 - M H z t r a n s d u c e r .

T w o - d i m e n s i o n a l a n d M - m o d e e c h o c a r d i o g -

r a p h y i n p a r a s t e r n a l l o n g - a n d s h o r t - a x i s

v i e w s w e r e p e r f o r m e d b l i n d e d a s p r e v i o u s l y

d e s c r i b e d a n d r e c o r d e d f o r p o s t e r i o r b l i n d e d

a n a l y s i s ( 3 ) .

R E S U L T S

CnAb 1 OVEREXPRESSION REDUCES CARDIAC

HYPERTROPHY. T o d e t e r m i n e t h e e f f e c t o f

C n Ab 1 o v e r e x p r e s s i o n o n t h e h e a r t i n t h e

c o n t e x t o f m a l a d a p t i v e c ar d i a c h y p e r t r o p h y ,

w e u s e d aM H C - C n Ab 1 t r a n s g e n i c m i c e t h a t o v e r -

e x p r e s s C n Ab 1 i n a c a r d i a c - s p e c i fi c m a n n e r ( O n l i n e

F i g u r e s 1 A a n d 1 B ) ( 4 ) . W e i n d u c e d p r e s s u r e o v e r -

l o a d i n w i l d - t y p e ( W T ) a n d t r a n s g e n i c m i c e b y T A C ,

a n d w e a n a l y z e d c a r d i a c f u n c t i o n 2 1 d a y s l a t e r .

T r a n s g e n i c m i c e s h o w e d a s i g n i fic a n t l y r e d u c e d h e a r t

w e i g h t t o b o d y w e i g h t r a t i o a f t e r T A C c o m p a r e d w i t h

W T m i c e ( F i g u r e 1 A ) . S i m i l a r l y , e c h o c a r d i o g r a p h i c

a n a l y s i s r e v e a l e d a r e d u c e d l e f t v e n t r i c u l a r m a s s i n -

d e x ( L V M i ) a n d t h i n n e r p o s t e r i o r w a l l a n d i n t e r v e n -

t r i c u l a r s e p t u m i n C n Ab 1 - o v e r e x p r e s s i n g m i c e a f t e r

T A C c o m p a r e d w i t h W T m i c e ( ) . I nF i g u r e s 1 B – 1 D

a g r e e m e n t w i t h t h e e c h o c a r d i o g r a p h y r e s u l t s , t r a n s -

g e n i c m i c e s h o w e d a m o r e l i m i t e d i n c r e a s e i n c a r -

d i o m y o c y t e s i z e ( F i g u r e 1 E ) . I m p o r t a n t l y , w h e r e a s

c o n t r a c t i l i t y d e c l i n e d i n W T m i c e 2 1 d a y s a f t e r T A C , i t

w a s p r e s e r v e d i n C n Ab 1 - o v e r e x p r e s s i n g m i c e , a s

s h o w n b y im p r o v e d l e f t v e n t r i c u l a r e j e c t i o n f r a c t i o n

( L V E F ) ( F i g u r e 1 F ) . T r a n s g e n i c m i c e a l s o s h o w e d a

l i m i t e d i n c r e a s e i n t h e e x p r e s s i o n o f t h e H F m a r k e r s

a t r i a l n a t r i u r e t i c fa c t o r ( A N F ) a n d b r a i n n a t r i u r e t i c

p e p t i d e ( B N P ) , w h i c h w e r e s t r o n g l y i n d u c e d i n W T

m i c e ( O n l i n e F i g u r e s 1 C a n d 1 D ) .

P r e s s u r e o v e r l o a d h y p e r t r o p h y w a s c h a r a c t e r i z e d

b y b o t h i n t e r s t i t i a l a n d p e r i v a s c u l a r fi b r o s i s i n W T

m i c e ( F i g u r e s 1 G t o 1 J ) . T r a n s g e n i c m i c e s h o w e d

s i g n i fi c a n t l y r e d u c e d c a r d i a c fi b r o s i s w i t h l e v e l s

s i m i l a r t o t h o s e o f s h a m - o p e r a t e d m i c e . I n a g r e e m e n t

S E E P A G E 6 6 8

A B B R E V I A T I O N S

A N D A C R O N Y M S

ATF4 = a c t i v a t i n g

t r a n s c r i p t i o n f a c t o r 4

BSO = L - b u t h i o n i n e -

s u l f o x i m i n e

GSH = r e d u c e d g l u t a t h i o n e

LVEF = l e f t v e n t r i c ul a r

e j e c t i o n f r a c t i o n

LVMi = l e f t v e n t r i c u l a r m a s s

i n d e x

mTOR = m e c h a n i s t i c t a r g e t o f

r a p a m y c i n

NFAT = n u c l e a r f a c t o r o f

a c t i v a t e d T c e l l s

qRT-PCR = q u a n t i t a t i v e

r e v e r s e - t r a n s c r i p t i o n r e a l - t i m e

p o l y m e r a s e c h a i n r e a c t i o n

TAC = t r a n s a o r t i c c o n s t r i c t i o n

WT = w i l d - t y p e

All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Padrón-

Barthe, Villalba-Orero, and Gómez-Salinero contributed equally to this work and are joint rst authors. Robyn Shaw, MD, PhD,fi

served as Guest Editor for this paper.

Manuscript received July 23, 2017; revised manuscript received November 16, 2017, accepted November 28, 2017.

J A C C V O L . 7 1 , N O . 6 , 2 0 1 8 Padrón-Barthe .et al

F E B R U A R Y 1 3 , 2 0 1 8 : 6 5 4 – 6 7 CnAb 1 Improves Function Through 1C Metabolism

655

https://doi.org/10.1016/j.jacc.2017.11.067




F I G U R E 1 b

Continued on the next page

Padrón-Barthe .et al J A C C V O L . 7 1 , N O . 6 , 2 0 1 8

CnAb 1 Improves Function Through 1C Metabolism F E B R U A R Y 1 3 , 2 0 1 8 : 6 5 4 – 6 7

656

w i t h t h e s e r e s u l t s , w e a l s o o b s e r v e d r e d u c e d e x p r e s -

s i o n o f t h e fi b r o s i s m a r k e r s c o l l a g e n Ia1 ( Co l 1a 1) a n d

l y s y l o x i d a s e ( L o x ) i n C n Ab 1 - o v e r e x p r e s s i n g m i c e ,

t o g e t h e r w i t h r e d u c e d l e v e l s o f S M A D 2 p h o s p h o r y l a -

t i o n , s u g g e s t i n g r e d u c e d T G F -b s i g n a l i n g ( O n l i n e

F i g u r e s 1 E t o 1 G ) .

T o c o n fir m t h e f u n c t i o n a l i m p r o v e m e n t e l i c i t e d b y

C n Ab 1 i n a s e c o n d t r a n s g e n i c m o u s e l i n e , w e u s e d

r t T A - C n Ab1 m i c e i n w h i c h C n Ab1 e x p r e s s i o n i s

i n d u c e d 3 - f o l d u p o n t r e a t m e n t w i t h d o x y c y c l i n e

( 3 ) . W e f o u n d t h a t C n Ab 1 o v e r e x p r e s s i o n s i g n i fi c a n t l y

r e d u c e d t h e l e f t v e n t r i c u l a r m a s s a n d i m p r o v e d s y s -

t o l i c f u n c t i o n 4 2 d a y s a f t e r T A C , c o n fir m i n g t h e r e -

s u l t s o b t a i n e d i n aM H C - C n Ab 1 m i c e ( O n l i n e F i g u r e 2 ) .

T o g e t h e r , t h e s e r e s u l t s d e m o n s t r a t e t h a t C n Ab 1 , u n -

l i k e o t h e r c a l c i n e u r i n i s o f o r m s , r e d u c e s m a l a d a p t i v e

h y p e r t r o p h y a n d c a r d i a c r e m o d e l i n g f o l l o w i n g p r e s -

s u r e o v e r l o a d .

C n Ab 1 O V E R E X P R E S S I O N D O E S N O T I N T E R F E R E

W I T H N F A T A C T I V A T I O N . T o d e t e r m i n e w h e t h e r

C n Ab 1 o v e r e x p r e s s i o n i n t e r f e r e s w i t h t h e a c t i v a t i o n

o f t h e e n d o g e n o u s N F A T p a t h w a y , w e fir s t q u a n t i fie d

t h e e x p r e s s i o n o f R c a n 1 i s o f o r m s . T A C i n d u c e d a

s i g n i fic a n t i n c r e a s e o f t h e R c a n 1 . 4 i s o f o r m , w h i c h i s

s t r o n g l y r e g u l a t e d b y N F A T , b u t n o t t h e R c a n 1 . 1 i s o -

f o r m . N o d i f f e r e n c e i n t h e e x p r e s s i o n o f R c a n 1 . 4 w a s

o b s e r v e d b e t w e e n W T a n d aM H C - C n Ab 1 m i c e

(F i g u r e s 2 A a n d 2 B ) . T o c o n fir m t h e a c t i v a t i o n o f t h e

N F A T p a t h w a y , w e c r o s s e d W T a n d aM H C - C n Ab1

m i c e w i t h a r e p o r t e r m o u s e l i n e i n w h i c h t h e

e x p r e s s i o n o f l u c i f e r a s e i s c o n t r o ll e d b y N F A T b i nd -

i n g s i t e s . A s t r o n g N F A T a c t i v a t i o n w a s o b s e r v e d i n

b o t h W T a n d t r a n s g e n i c m i c e i n r e s p o n s e t o p r e s s u r e

o v e r l o a d , a s d e t e r m i n e d b y l u c i f e r a s e a c t i v i t y ,

w i t h o u t a n y s u b s t a n t i a l c h a n g e b e t w e e n b o t h m o u s e

t y p e s ( F i g u r e 2 C ) . T h e s e r e s u l t s i n d i c a t e t h a t C n Ab1

o v e r e x p r e s s i o n d o e s n o t i n t e r f e r e w i t h N F A T a c t i v a -

t i o n i n c a r d i a c h y p e r t r o p h y .

C n Ab 1 A C T I V A T E S S E R I N E A N D O N E - C A R B O N

M E T A B O L I S M . W e n e x t a n a l y z e d t h e t r a n s c r i p t i o n

p r o fi l e o f W T a n d t r a n s g e n i c m i c e u s i n g m i c r o a r r a y s .

F o l l o w i n g T A C , W T m i c e s h o w e d a c o n s i s t e n t

u p - r e g u l a t i o n o f g e n e s a s s o c i a t e d w i t h e x t r a c e l l u l a r

m a t r i x , c e l l a d h e s i o n , a n d fi b r o s i s ( O n l i n e T a b l e s 1

a n d 2 ) a n d a d o w n - r e g u l a t i o n o f g e n e s a s s o c i a t e d

w i t h t h e m i t o c h o n d r i a ( O n l i n e T a b l e s 3 a n d 4 ) .

I n t e r e s t i n g l y , s h a m aM H C - C n Ab 1 m i c e s h o w e d a

s t r o n g u p - r e g u l a t i o n o f t h e g e n e s i n v o l v e d i n s e r i n e

s y n t h e s i s , p h o s p h o g l y c e r a t e d e h y d r o g e n a s e ( P h g d h ) ,

a n d p h o s p h o s e r i n e a m i n o t r a n s f e r a s e 1 ( P s a t 1 ) , a n d i n

t h e o n e - c a r b o n p a t h w a y , i n c l u d i n g m e t h y l t e t r a h y -

d r o f o l a t e d e h y d r o g e na s e 2 ( M t h f d 2 ) a n d a l d e h y d e

d e h y d r o g e n a s e 1 L 2 ( A l d h 1 l 2 ) ( F i g u r e 2 D , O n l i n e

T a b l e s 5 t o 8 ) . U s i n g q u a n t i t a t i v e r e v e r s e -

t r a n s c r i p t i o n r e a l - t i m e p o l y m e r a s e c h a i n r e a c t i o n

( q R T - P C R ) , w e c o n fi r m e d t h e s i g n i fi c a n t i n d u c t i o n o f

P h g d h , P s a t 1 , S h m t 2 , M t h f d 2 , a n d A l d h 1 l 2 g e n e s i n

aM H C - C n Ab 1 m i c e , a f t e r b o t h s h a m a n d T A C s u r g e r y

(F i g u r e s 2 E t o 2 I ) . I n a d d i t i o n , m o s t o f t h e s e g e n e s , a s

w e l l a s C n Ab 1 i t s e l f , s h o w e d s m a l l e r , b u t s i g n i fi c a n t ,

e x p r e s s i o n c h a n g e s i n W T m i c e f o l l o w i n g T A C ( O n l i n e

F i g u r e 3 ) , s u g g e s t i n g t h a t e n d o g e n o u s C n Ab1 a n d t h e

s e r i n e a n d o n e - c a r b o n p a t h w a y m a y p l a y a r o l e i n t h e

r e s p o n s e t o p r e s s u r e o v e r l o a d .

A l t h o u g h n o g e n e c a t e g o r i e s c h a n g e d s i g n i fi c a n t l y

i n t r a n s g e n i c m i c e a f t e r T A C ( On li ne T ab le s 9 to 1 2) ,

aM H C - C n Ab 1 m i c e s h o w e d r e d u c e d e x p r e s s i o n o f

e x t r a c e l l u l a r – m a t r i x - r e l a t e d g e n e s c o m p a r e d w i t h

W T ( O n l i n e T a b l e s 1 3 t o 1 6 ) , w h i c h w a s c o n fi r m e d b y

q R T - P C R ( O n l i n e F i g u r e s 1 E a n d 1 F ) .

W e t h e n c a r r i e d o u t a q u a n t i t a t i v e p r o t e o m i c s

a n a l y s i s o f s h a m W T a n d t r a n s g e n i c m i c e , w h i c h a l s o

s h o w e d a s t r o n g i n c r e as e i n p r o t e i n s i n v o l v e d i n t h e

s e r i n e a n d o n e - c a r b o n p a t h w a y i n aM H C - C n Ab 1 m i c e

(O n l i n e T a b l e 1 7 , F i g u r e 2 D ) , c o n fi r m i n g t h a t s e r i n e

a n d on e - c a r b o n m e t a b o l is m w a s s t r o n g l y i n d u c e d i n

t h e h e a r t b y C n Ab1 .

M a n y o f t h e s e g e n e s a r e r e g u l a t e d b y t h e m T O R

s i g n a l i n g p a t h w a y t h r o u g h t h e t r a n s c r i p t i o n f a c t o r

a c t i v a t i n g t r a n s c r i p t i o n f a c t o r 4 ( A T F 4 ) ( 4 , 1 0 ) . T o

d e t e r m i n e w h e t h e r t h i s p a t h w a y w a s a c t i v a t e d

b y C n Ab 1 i n t h e c o n t e x t o f p r e s s u r e o v e r l o a d , w e

a n a l y z e d t h e s e p r o t e i n s b y W e s t e r n b l o t . W e o b s e r v e d

a c t i v a t i o n o f t h e m T O R / A K T s i g n a l i n g p a t h w a y a n d

i n c r e a s e d e x p r e s s i o n o f A T F 4 i n C n Ab1 - o v e r e x p r e s s i n g

F I G U R E 1

(A) Pressure overload cardiac hypertrophy was induced in wild type (WT) and aMHC-CnAb 1 mice by transaortic constriction (TAC) and the heart weight to

body weight ratio was determined 21 days after surgery, compared with sham-operated animals. LVMi , LVPWd , and IVSd were determined by(B) (C) (D)

echocardiography 21 days after surgery. Cardiomyocyte cross-sectional area was analyzed by immunohistochemistry. LVEF was determined by(E) (F)

echocardiography. Interstitial and perivascular cardiac brosis were analyzed 21 days after TAC using Masson s trichrome protocol.(G and H) (I and J) fi ’

Bar indicates 100 m m. Interstitial brosis was quanti ed as the percentage of area in each picture that is stained as collagen. Perivascular brosis wasfi fi fi

calculated as the perivascular collagen area relative to the vessel area. At least 2 pictures per mouse were analyzed. Values represent average SEM.

**p 0.01, ***p 0.005 TAC versus sham for each mouse line; p 0.05, p 0.01, p 0.005 WT versus transgenic mice; 2-way analysis† †† †††

of variance plus Bonferroni post-test. n 12 to 18. CSA cross-sectional area; HW/BW heart weight to body weight ratio; IVSd interventricular¼ ¼ ¼ ¼

septum thickness in diastole; LVEF left ventricular ejection fraction; LVMi left ventricular mass index; LVPWd left ventricular posterior wall¼ ¼ ¼

thickness in diastole.



657

F I G U R E 2 b

Expression of the Rcan1.4 and Rcan1.1 mRNA was analyzed by qRT-PCR 21 days after TAC. WT and(A) (B) (C) aMHC-CnAb 1 mice were crossed with reporter mice in

which luciferase expression is controlled by an NFAT binding site multimer. Luciferase activity normalized to total protein content was analyzed in heart extracts

21 days post-TAC. Summary of genes involved in the serine and one-carbon pathway. Components induced in(D) aMHC-CnAb 1 mice are indicated by a (mRNA,blue ll

metabolites) or (protein). Expression of Phgdh , Psat1 , Shmt2 , Mthfd2 , and Aldh1l2 was quanti ed as in . Values express averagepurple border (E) (F) (G) (H) (I) fi (A)

SEM. *p 0.05, **p 0.01 TAC versus sham for each mouse line; p 0.05, p 0.01, p 0.005 WT versus transgenic mice; 2-way analysis of variance plus† †† †††

Bonferroni post-test. n 10 to 15¼ (A, B, and E I)– ; n ¼ 5 to 12 (C). 3PG 3-phosphoglycerate; 3P-Ser 3-phosphoserine; Gly glycine; NAD(P) nicotinamide adenine¼ ¼ ¼ ¼

dinucleotide phosphate; NAD(P)H nicotinamide adenine dinucleotide phosphate (reduced form); NFAT nuclear factor of activated T cells; qRT-PCR quantitative¼ ¼ ¼

reverse-transcription real-time polymerase chain reaction; Ser serine; THF tetrahydrofolate; other abbreviations as in .¼ ¼ Figure 1



658

m i c e ( F i g u r e 3 A ) , c o n s i s t e n t w i t h o u r p r e v i o u s r e s u l t s

( 3 , 4 ) . T o i n v e s t i g a t e w h e t h e r m T O R m e d i a t e s t h e

i n d u c t i o n o f t h e s e r i n e a n d o n e - c a r b o n p a t h w a y b y

C n Ab 1 , w e t r e a t e d u n i n j u r e d W T a n d t r a n s g e n i c m i c e

w i t h t h e m T O R i n h i b i t o r r a p a m y c i n , a n d w e a n a l y z e d

t h e m i c e 5 d a y s l a t e r . A s s h o w n i n F i g u r e s 3 B t o 3 D ,

m T O R i n h i b i t i o n p a r t i a l l y a b o l i s h e d t h e i n d u c t i o n o f

P h g d h , P s a t 1 , a n d M t h f d 2 i n aM H C - C n Ab 1 m i c e ,

i n d i c a t i n g t h a t C n Ab 1 a c t i v a t e s t h e s e r i n e a n d o n e -

c a r b o n p a t h w a y th r o u g h m T O R s i g n a l i ng .

C n Ab 1 O V E R E X P R E S S I O N C H A N G E S T H E H E A R T S

M E T A B O L O M E . P h d g h - d e r i v e d s e r i n e s y n t h e s i s a n d

t h e o n e - c a r b o n c y c l e s u p p l y m e t a b o l i t e s f o r t h e p r o -

d u c t i o n o f a n t i o x i d a n t m e d i a t o r s s u c h a s g l u t a t h i o n e

( 1 1 , 1 2 ) . A t h o r o u g h m e t a b o l o m i c a n a l y s i s i n u n i n j u r e d

W T a n d aM H C - C n Ab 1 m i c e d e t e c t e d a t o t a l o f

4 6 2 d i f f e r e n t m e t a b o l i t e s i n m y o c a r d i a l s a m p l e s

f r o m b o t h m o u s e t y p e s , w i t h 1 3 3 o f t h e m b e i n g u p -

r e g u l a t e d i n t r a n s g e n i c m i c e a n d 6 5 m e t a b o l i t e s

d o w n - r e g u l a t e d ( O n l i n e T a b l e 1 8 ) . A p r i n c i p a l

c o m p o n e n t a n a l y s i s s h o w e d s t r o n g s e p a r a t i o n o f

t h e s a m p l e s , s u g g e s t i n g a d i f f e r e n t m e t a b o l i c p r o fi l e

i n W T a n d C n Ab 1 - o v e r e x p r e s s i n g m i c e ( O n l i n e

F i g u r e 4 A ) . U s i n g M e t a b o l o m i c P a t h w a y A n a l y s i s , w e

f o u n d a s t r o n g e n r i c h m e n t of m e t a b o l i t e s r e l a t e d t o

g l u t a t h i o n e , a m i n o a c i d , a n d n u c l e o t i d e m e t a b o l i c

p a t h w a y s ( O n l i n e F i g u r e 4 B ) .

T o e x p l o r e t h e s e c h a n g e s m o r e i n d e p t h , w e

a n a l y z e d t h e a m o u n t o f d i f f e r e n t m e t a b o l i t e s

b e l o n g i n g t o t h e a f f e c t e d p a t h w a y s . W e f o u n d a

s t r o n g s i g n i fic a n t i n d u c t i o n o f g l u t a t h i o n e , m a i n l y

r e d u c e d g l u t a t h i o n e ( G S H ) , a n d i t s p r e c u r s o r m e t a b -

o l i t e s ( O n l i n e F i g u r e s 4 C a n d 5 , O n l i n e T a b l e 1 8 ) . T h i s

w a s a c c o m p a n i e d b y a s i g n i fic a n t i n d u c t i o n o f

m e t h i o n i n e m e t a b o l i t e s ( O n l i n e F i g u r e 4 D ) . I n a d d i -

t i o n , aM H C - C n Ab 1 m i c e s h o w e d a s i g n i fi c a n t i n c r e a s e

o f s e v e r a l m e t a b o l i t e s a s s o c i a t e d w i t h p o l y a m i n e

m e t a b o l i s m a n d t h e u r e a c y c l e , m a i n l y r e l a t e d t o t h e

d e g r a d a t i o n o f a r g i n i n e t o o r n i t h i n e , p u t r e s c i n e , a n d

s p e r m i d i n e ( O n l i n e T a b l e 1 8O n l i n e F i g u r e 4 E , ) . W e

a l s o f o u n d t h a t s e v e r a l m e t a b o l i t e s r e l a t e d t o p u r i n e

a n d p y r i m i d i n e m e t a b o l i s m w e r e u p - r e g u l a t e d ,

l e a d i ng t o a s t r o n g i n d u c ti o n o f t h e p o t e n t a n t i ox i -

d a n t u r a t e ( O n l i n e T a b l e 1 8 ) . T h e s e c h a n g e s w e r e

F I G U R E 3 b

(A) Phosphorylation and/or expression of mTOR, Akt, and ATF4 was analyzed by Western blot in myocardial samples from the indicated mice

21 days after surgery. WT and(B to D) aMHC-CnAb 1 uninjured mice were treated with rapamycin for 5 days, and the expression of Phgdh,

Psat1, and Mthfd2 was analyzed in myocardial samples. *p 0.05, ***p 0.005 aMHC-CnAb 1 versus WT; p 0.01, p 0.005†† †††

rapamycin versus vehicle; 2-way analysis of variance plus Bonferroni post-test. n 5. ATF4 activating transcription factor 4;¼ ¼

mTOR mechanistic target of rapamycin; other abbreviations as in .¼ Figure 1



659

p a r a l l e l e d b y a s t r o n g i n c r e a s e i n a n o t h e r a n t i o x i -

d a n t , a s c o r b a t e ( O n l i n e F i g u r e 4 F ) . T o g e t h e r , t h e s e

c h a n g e s i n d i c a t e d t h a t C n Ab1 a c t i v a t e s s ev er a l

m e t a b o l i c p a t h w a y s w i t h a n t i o x i d a n t a c t i v i t y .

M I T O C H O N D R I A L C O M P L E X E S S H O W I N C R E A S E D

A C T I V I T Y I N C n Ab 1 - O V E R E X P R E S S I N G M I C E . T o

d et er m i n e w h e t h e r t h e c h a n g e s i n t h e m e t a b o l i c

p r o fil e o b s e r v e d i n t r a n s g e n i c m i c e w o u l d i m p a c t

m i t o c h o n d r i a l n u m b e r o r a c t i v i t y , w e a n a l y z e d

d i f f e r e n t p a r a m e t e r s i n u n i n j u r e d m i c e . W e fi r s t

q u a n t i fie d m i t o c h o n d r i a l D N A a n d f o u n d n o s i g n i fi-

c a n t d i f f e r e n c e s b e t w e e n W T a n d aM H C - C n Ab 1 m i c e ,

a l t h o u g h a s m a l l i n c r e a s e w a s o b s e r v e d i n t r a n s g e n i c

m i c e ( O n l i n e F i g u r e 6 A ) . A n a l y s i s o f m i t o c h o n d r i a l

c o m p l e x e s a n d s u p e r c o m p l e x e s i n i s o l a t e d m i t o -

c h o n d r i a r e v e a l e d n o d i f f e r e n c e s b e t w e e n b o t h m o u s e

t y p e s ( O n l i n e F i g u r e 6 B ) . S i m i l a r l y , w e o b s e r v e d n o

o b v i o u s d i f f e r e n c e s i n t h e e x p r e s s i o n o f i n d i v i du a l

c o m p l e x c o m p o n e n t s b y W e s t e r n b l o t ( O n l i n e

F i g u r e 6 C ) . A n a l y s i s o f t h e a c t i v i t y o f t h e d i f f e r e n t

m i t o c h o n d r i a l c o m p l e x e s s h o w e d a s i g n i fi c a n t i n -

c r e a s e i n t h e a c t i v i t y o f c o m p l e x e s I þI I I a n d I I þI I I i n

aM H C - C n Ab 1 m i c e ( O n l i n e F i g u r e 6 D ) . I n a d d i t i o n , w e

o b s e r v e d i n c r e a s e d c i t r a t e s y n t h a s e a c t i v i t y i n t r a n s -

g e n i c m i c e , i n a g r e e m e n t w i t h t h e i n c r e a s e i n c i t r a t e

d e t e c t e d b y m a s s s p e c t r o m e t r y ( O n l i n e T a b l e 1 8 ) an d

w i t h t h e s l i g h t i n c r e a s e i n m t D N A c o p y n u m b e r

( m i t o c h o n d r i a l m a s s ) . T h e s e c h a n g e s i n t h e a c t i v i t y o f

m i t o c h o n d r i a l c o m p l e x e s , h o w e v e r , h a d n o e f f e c t o n

t h e p r o d u c t i o n o f A T P b y i s o l a t e d m i t o c h o n d ri a ,

n e i t h e r d r i v e n b y g l u t a m a t e a n d m a l a t e n o r b y s u c c i -

n a t e ( O n l i n e F i g u r e 6 E ) .

CnAb 1 REDUCES PROTEIN OXIDATION AND IMPROVES ATP

SYNTHESIS F OLLOW ING PRESSURE OVERLOAD . W e n e x t

i n v e s t i g a t e d w h e t h e r t h e c h a n g e s o b s e r v e d i n u n i n -

j u r e d m i c e w e r e r e p r o d u c e d i n t h e h y p e r t r o p h i c

F I G U R E 4 b

Reduced and total glutathione, and ATP generation driven by glutamate malate or by succinate were quanti ed in whole myocardium from WT and(A) (B) þ (C) (D) fi

aMHC-CnAb 1 mice 21 days after TAC or sham surgery. *p 0.05, **p 0.01 versus sham; p 0.01 WT versus transgenic mice; 2-way analysis of variance plus††

Bonferroni post-test. n ¼ 9 t o 15 8 to 14(A and B), n ¼ (C and D) (E and F). Quantitative redox proteomics analysis using the GELSILOX technique. Shown are the

cumulative distributions of the standardized log 2 ratios of abundances (Z qp ) of peptides containing oxidized cysteine residues in the conditions that are compared. The

peptides belong to either mitochondrial proteins or proteins associated with the Krebs cycle or muscle contraction . The null hypothesis distribution, which(E) (F)

re ects identical peptide abundances in the 2 conditions, is represented by a . The show the relative abundance of peptides from WT mice after TAC infl gray line blue lines

relation to those from WT mice after sham surgery, whereas the represent the peptides fromorange lines aMHC-CnAb 1 mice after TAC in relation to WT mice after TAC.

Positive values of Zqp indicate increase in relative peptide abundance. ATP adenosine triphosphate; GSH reduced glutathione; other abbreviations as in .¼ ¼ Figure 1



660

h e a r t . U s i n g a n e n z y m a t i c a s s a y , w e f o u n d a n i n -

c r e a s e i n G S H i n aM H C - C n Ab 1 m i c e c o m p a r e d w i t h

W T , w h i c h w a s m o r e e v i d e n t 2 1 d a y s a f t e r T A C

(F i g u r e 4 A ) . W e a l s o f o u n d a n i n c r e a s e i n t o t a l g l u t a -

t h i o n e i n t r a n s g e n i c m i c e , a l b e i t a t m u c h l o w e r l e v e l s

(F i g u r e 4 B ) . W e o b s e r v e d a s t r o n g r e d u c t i o n i n t h e

p r o d u c t i o n o f A T P b y m i t o c h o n d r i a i s o l a t e d f r o m W T

m i c e a f t e r T A C , b o t h d r i v e n b y g l u t a m a t e a n d m a l a t e

(F i g u r e 4 C ) a n d b y s u c c i n a t e ( F i g u r e 4 D ) . B y c o n t r a s t ,

m i t o c h o n d r i a f r o m aM H C - C n Ab 1 m i c e s h o w e d p r e -

s e r v e d A T P p r o d u c t i o n l e v e l a f t e r T A C , s i g n i fic a n t l y

h i g h e r t h a n t h a t o f W T m i c e ( F i g u r e s 4 C a n d 4 D ) . U s -

i n g t h e G E L S I L O X t e c h n i q u e ( 1 3 ) , w e o b s e r v e d a n

i n c r e a s e i n o x i d i z e d p e p t i d e s f r o m m i t o c h o n d r i a l

p r o t e i n s , p a r t i c u l a r l y t h o s e r e l a t e d t o t h e K r e b s c y c l e ,

a n d f r o m m u s c l e c o n t r a c t i o n p r o t e i n s i n W T m i c e

2 1 d a y s a f t e r T A C ( W T - T A C m i c e ) ( F i g u r e s 4 E a n d 4 F ,

O n l i n e T a b l e s 1 9 t o 2 1 ) . I n t e r e s t i n g l y , aM H C - C n Ab1

m i c e s u b j e c t e d t o T A C s h o w e d r e d u c e d o x i d a t i o n o f

t h e s a m e p e p t i d e p o p u l a t i o n s i n c om p a r i s o n t o W T -

T A C m i c e ( F i g u r e s 4 E a n d 4 F ) , s u g g e s t i n g t h a t t h e

a n t i o x i d an t r e s p o n s e e l i c i t e d b y C n Ab1 p r ev en t s

m i t o c h o n d r i a l a n d s a r c o m e r e p r o t e i n o x i d a t i o n , a n d

h e n c e i m p r o v e s A T P p r o d u c t i o n a n d c a r d i a c

c o n t r a c t i o n .

A C T I V A T I O N O F T H E S E R I N E A N D O N E - C A R B O N

P A T H W A Y I S N E C E S S A R Y F O R T H E I N D U C T I O N O F

A T P A N D R E D U C T I O N O F C A R D I A C H Y P E R T R O P H Y

B Y C n Ab 1 . T o d e t e r m i n e t h e f u n c t i o n a l r e l e v a n c e o f

t h e a c t i v a t i o n o f s e r i n e o n e - c a r b o n m e t a b o l i s m b y

C n Ab 1 , w e t r e a t e d W T a n d t r a n s g e n i c m i c e f o r 2 1 d a y s

a f t e r T A C s u r g e r y w i t h t h e P H G D H i n h i b i t o r N C T - 5 0 3 ,

w h i c h b l o c k s t h e s y n t h e s i s o f s e r i n e f r o m g l u c o s e a n d

i t s c o n t r i b u t i o n t o t h e o n e - c a r b o n p a t h w a y ( 1 4 ) .

T r e a t m e n t o f aM H C - C n Ab 1 m i c e w i t h N C T - 5 0 3 r e su l -

t e d i n i n c r e a s e d L V M i a n d r e d u c e d L V E F c o m p a r e d

w i t h u n t r e a t e d t r a n s g e n i c m i c e ( F i g u r e s 5 A a n d 5 B ) .

F u r t h e r m o r e , N C T - 5 0 3 p r e v e n t e d t h e i m p r o v e d A T P

F I G U R E 5 b

Mice underwent TAC or sham surgery and LVMi and LVEF were analyzed by echocardiography 21 days later. Where indicated, mice(A) (B)

were treated with the PHGDH inhibitor NCT-503 for 21 days. ATP synthesis from glutamate and malate or succinate was analyzed in all(C) (D)

mice. Note that the control (untreated) animals for these analyses are those shown in (for and ) and (for and ), and areFigure 1 A B Figure 6 C D

repeated here for comparative purposes. *p 0.05, **p 0.01, ***p 0.005 TAC versus sham for each mouse line; p 0.01, p†† †††

0.005 WT versus transgenic mice; p 0.05, TAC NCT-503 treated mice versus TAC untreated mice; 2-way analysis of variance plus‡ –

Bonferroni post-test. n ¼ 6 to 18 6 to 14(A and B), n ¼ (C and D). Abbreviations as in Figures 1 and 4.



661

s y n t h e s i s i n d u c e d b y C n Ab 1 , b o t h f r o m g l u t a m a t e a n d

m a l a t e , a n d f r o m s u c c i n a t e . T r a n s g e n i c m i c e s h o w e d

s i m i l a r A T P l e v e l s t o t h o s e o f W T m i c e a n d s i g n i fi -

c a n t l y r e d u c e d c o m p a r e d w i t h u n t r e a t e d aM H C -

C n Ab 1 m i c e ( F i g u r e s 5 C a n d 5 D ) . T h e s e d a t a i n d i c a t e

t h a t t h e se r i n e a n d o n e - ca r b o n p a t h w a y i s n e c e s s a r y

f o r t h e b e n e fic i a l e f f e c t s o f C n Ab 1 o n t h e h e a r t .

GLUTATHIONE MEDIATES THE IMPROVED RESPONSE TO

PRESSURE OVERLOAD IN MICE. T o fi n d o u t

w h e t h e r t h e i n c r e a s e i n r e d u c e d g l u t a t h i o n e w a s a l s o

r e s p o n s i b l e f o r t h e i m p r o v e m e n t i n A T P p r o d u c t i o n

a n d c a r d i a c f u n c t i o n o b s e r v e d i n aM H C - C n Ab 1 m i c e

a f t e r T A C , w e t r e a t e d b o t h W T a n d C n Ab1 o v e r -

e x p r e s s i n g m i c e w i t h B S O , w h i c h d e c r e a s e s g l u t a -

t h i o n e s y n t h e s i s b y i n h i b i t in g g- g l u t a m y l c y s t e i n e

s y n t h e t a s e ( 1 5 ) . A d m i n i s t r a t i o n o f B S O r e s u l t e d i n a

s t r o n g r e d u c t i o n i n b o t h G S H a n d t o t a l g l u t a t h i o n e i n

b o t h W T a n d t r a n s g e n i c m i c e , a n d p r e v e n t e d t h e i n -

c r e a s e i n G S H i n d u c e d b y C n Ab 1 ( F i g u r e s 6 A a n d 6 B ) .

T h e l e v e l s o f u n t r e a t e d s h a m - o p e r a t e d W T m i c e a r e

s h o w e d f o r r e f e r e n c e . I n h i b i t i o n o f g l u t a t h i o n e s y n -

t h e s i s c a u s e d a s t r o n g d e c r e a s e i n A T P s y n t h e s i s i n a l l

m i c e ( F i g u r e s 6 C a n d 6 D ) , c o mp a r e d w it h u n t r ea t e d

s h a m W T m i c e . T h i s w a s a c c o m p a n i e d b y a s l i g h t

r e d u c t i o n i n v e n t r i c u l a r w a l l t h i c k n e s s , a l t h o u g h

c a r d i a c h y p e r t r o p h y p r o c e e d e d n o r m a l l y i n r e sp o n s e

t o T A C ( F i g u r e 6 E ) . I m p o r t a n t l y , i n t h e p r e s e n c e o f

B S O , W T a n d aM H C - C n Ab 1 m i c e s h o w e d a s i m i l a r

i n c r e a s e i n L V M i ( F i g u r e 6 E ) . B S O t r e a t m e n t a l s o

r e s u l t e d i n r e d u c e d L V E F f o l l o w i n g T A C i n C n Ab1 -

o v e r e x p r e s s i n g m i c e , w h e r e a s n o d e c l i n e i n c a r d i a c

c o n t r a c t i l i t y w a s o b s e r v e d i n W T m i c e ( F i g u r e 6 F ) .

T o g e t h e r , t h e s e r e s u l t s s u g g e s t t h a t t h e i n d u c t i o n o f

G S H b y C n Ab 1 i s n e c e s s a r y f o r t h e f u n c t i o n a l a n d

s t r u c t u r a l i m p r o v e m en t t r i g g e r e d b y t h i s c a l c i n e u r i n

i s o f o r m a n d t h a t i n t h e a b s e n ce o f g l u t a t h i on e , t h e

i n c r e a s e d s u b s t r a t e flu x t h r o u g h t h e o x i d a t i ve p h o s -

p h o r y l a t i o n c h a i n i n d u c e d b y C n Ab1 m a y ha v e d e l e -

t e r i o u s e f f e c t s o n c a r d i a c f u n c t i o n d e c o m p e n s a t i o n

f o l l o w i n g p r e s s u r e o v e r l o a d .

MICE LACKING CnAb 1 DEVELOP CARDIAC HYPERTROPHY.

T o d e t e r m i n e t h e r o l e o f e n d o g e n o u s C n Ab 1 , w e

d e v e l o p e d k n o c k o u t m i c e l a c k i n g i n t r o n 1 2 - 1 3 i n t h e

C n Ab g e n e ( C n Ab1D i 1 2 m i c e ) , w h i c h c o d e s f o r t h e

F I G U R E 6 b

(A and B) (A) (B)WT and transgenic mice underwent sham or TAC surgery and were treated with L-buthionine-sulfoximine (BSO) for 21 days. Reduced and total

glutathione, and ATP synthesis from glutamate and malate or succinate were measured in myocardial samples. Values are expressed as percentage SEM,(C) (D)

referred to untreated WT sham mice . LVMi and LVEF were determined by echocardiography in BSO-treated and untreated (control) mice 21 days(dashed line) (E) (F)

after surgery. Note that the control (untreated) animals for these comparisons are those shown in , and are repeated here for comparative purposes. *p 0.05,Figure 1

**p 0.01, ***p 0.005, TAC versus sham for each mouse line; p 0.05, p 0.005† ††† aMHC-CnAb 1 versus WT; p 0.05 BSO versus control; 2-way analysis of‡

variance plus Bonferroni post-test. n ¼ 5 to 9 (A and B), n ¼ 3 to 9 (C and D), n ¼ 5 to 18 (E and F). Abbreviations as in Figures 1 and 4.



662

F I G U R E 7 b

(A) Schematic of the strategy used to generate the CnAbDi12 mice. CnA(B and C) b 1 and CnAb 2 expression was analyzed by qRT-PCR in WT and CnAb

Di12 mice. n 19 to¼

25. The corrected and indexed left ventricular (LV) mass were analyzed by echocardiography at 2, 8, and 15 months of age. n 6 to 18. Cardiomyocyte(D) (E) ¼ (F)

cross-sectional area was analyzed by histological methods. n 5 to 14. Systolic and diastolic blood pressure were measured in 15-month-old WT and¼ (G) CnAbDi12 mice.

n 6 to 11. LVEF was analyzed by echocardiography at 2, 8, and 15 months. n 7 to 18. ATP synthesis from glutamate and malate or from succinate was¼ (H) ¼ (I) (J)

quanti ed in WT andfi CnAbDi12

mice. n 6 to 10. BNP and Rcan1.4 mRNA expression was quanti ed by qRT-PCR. n 19 to 25. Western blot analysis of¼ (K and L) fi ¼ (M)

NFATc3 in WT and CnAb1 Di12 mice (n 4) (smaller bands indicate activation by dephosphorylation). Phosphorylated (active) and total forms of kinases in the p38 and¼

JNK pathways were also analyzed. **p 0.01, ***p 0.005, 8 and 15 months versus 2 months for each mouse line; p 0.05, p 0.01, WT versus† †† CnA bDi12 mice;

2-way analysis of variance plus Bonferroni post-test. Abbreviations as in .Figures 1 and 4



663

u n i q u e C - t e r m i n a l d o m a i n i n C n Ab 1 ( F i g u r e 7 A ) .

A l t h o u g h t h e s e m i c e e x p r e s s n o C n Ab 1 , e x p r e s s i o n

o f t h e t r a d i t i o n a l i s o f o r m C n Ab 2 i s u n a l t e r e d

(F i g u r e s 7 B a n d 7 C ) . C n Ab1Di 1 2 m i c e a r e v i a b l e a n d

f e r t i l e , a n d a r e b o r n a t t h e e x p e c t e d M e n d e l i a n r a t i o .

H o w e v e r , w e o b s e r v e d a s i g n i fic a n t i n c r e a s e i n c a r -

d i a c h y p e r t r o p h y w i t h a g e . E c h o c a r d i o g r a p h i c a n a l -

y s i s s h o w e d i n c r e a s e d l e f t v e n t r i c u l a r m a s s i n

C n Ab1Di 1 2 m i c e a t 1 5 m o n t h s o f a g e a n d i n c r e a s e d

c a r d i o m y o c y t e a r e a ( F i g u r e s 7 D t o 7 F ) . N o c h a n g e s

w e r e o b s e r v e d i n b l o o d p r e s s u r e ( F i g u r e 7 G ) , s u g -

g e s t i n g t h a t c a r d i a c h y p e r t r o p h y w a s n o t t h e

c o n s e q u e n c e o f s y s t e m i c h y p e r t e n s i o n . C a r d i a c h y -

p e r t r o p h y w a s a c c o m p a n i e d b y a d e c l i n e i n L V E F i n

C n Ab 1 - d e fi c i e n t m i c e ( F i g u r e 7 H ) . T h i s r e d u c e d

c o n t r a c t i l e c a p a c i t y w a s p a r a l l e l e d b y a d e c r e a s e d

a b i l i t y t o p r o d u c e A T P , b o t h f r o m g l u t a m a t e a n d

m a l a t e a n d f r o m s u c c i n a t e ( F i g u r e s 7 I a n d 7 J ) .

F u r t h e r m o r e , w e f o u n d i n c r e a s e d e x p r e s s i o n o f B N P

a n d t h e N F A T - r e g u l a t e d R c a n 1 . 4 i s o f o r m i n

C n A b1Di 1 2 m i c e ( F i g u r e s 7 K a n d 7 L ) . I n c r e a s e d a c t i -

v a t i o n o f N F A T c 3 w a s c o n fi r m e d b y W e s t e r n b l o t

(F i g u r e 7 M ) , a n d i t w a s l i k e l y d u e t o t h e i n h i b i t i o n o f

t h e M A P K s p 3 8 a n d J N K , w h i c h r e p h o s p h o r y l a t e a n d

i n h i b i t N F A T c . I n h i bi t i o n o f J N K a n d p 3 8 , t h e i r u p -

s t r e a m k i n a s e s M K K 4 a n d M K K 3 / 6 , o r t h e i r u p s t r e a m

M K K k i n a s e A S K 1 h a s b e e n r e p o r t e d t o i n d u c e c a r -

d i a c h y p e r t r o p h y t h r o u g h N F A T a c t i v a t i o n ( 1 6 –1 8 ) .

W e f o u n d d e c r e a s e d p h o s p h o r y l a t i o n ( i n d i c a t i n g

d e c r e a s e d a c t i v a t i o n ) o f t h e M K K 4 / J N K a n d t h e

F I G U R E 8 bD

Left ventricular (LV) mass corrected , LVMi , and LVEF were analyzed by echocardiography 21 days after TAC in WT and(A) (B) (C) CnAbD i12 mice. n ¼ 7 t o 8.

Expression of ANF , Phgdh , and Psat1 was analyzed by WT and(D) (E) (F) CnAbDi12 mice by qRT-PCR 21 days after surgery. n ¼ 5 to 7 . *p 0.05,

**p 0.01, ***p 0.005 TAC versus Sham for each mouse line; p 0.05, p 0.01, p 0.005, WT versus† †† ††† CnA bDi12 mice; 2-way analysis of variance

plus Bonferroni post-test. ANF atrial natriuretic factor; other abbreviations as in .¼ Figure 1



664

M K K 3 / 6 / p 3 8 p a t h w a y s a n d a m i l d i n h i b i t i o n o f A S K 1 ,

w h i c h r e g u l a t e s b o t h p a t h w a y s , i n C n Ab1Di 1 2 m i c e

(F i g u r e 7 M ) . T h e s e r e s u l t s s u g g e s t t h a t t h e i n c r e a s e d

h y p e r t r o p h y o b s e r v e d i n o l d C n Ab 1Di 1 2 m i c e i s t h e

r e s u l t o f J N K a n d p 3 8 i n h i b i t i o n a n d s u b s e q u e n t

N F A T c a c t i v a t i o n . W e n e x t s t u d i e d t h e r e s p o n s e o f

C n Ab1 Di 1 2 m i c e t o p r e s s u r e o v e r l o a d . T h r e e w e e k s

a f t e r T A C , C n Ab 1 - d e fi c i e n t m i c e s h o w e d i n c r e a s e d

c a r d i a c h y p e r t r o p h y , i n d i c a t e d b y t h e l a r g e r l e f t

v e n t r i c u l a r m a s s ( F i g u r e s 8 A a n d 8 B ) . L V E F , h o w e v -

e r , w a s h i g h e r i n C n Ab1D i 1 2 t h a n i n c o n t r o l m i c e

(F i g u r e 8 C ) , s u g g e s t i n g t h a t c a r d i a c h y p e r t r o p h y h a d

n o t y e t d e c o m p e n s a t e d . C n Ab1Di 1 2 m i c e s h o w e d

h i g h e r A N F e x p r e s s i o n a f t e r T A C s u r g e r y , i n a g r e e -

m e n t w i t h t h e i n c r e a s e d h y p e r t r o p h y , a n d r e d u c e d

e x p r e s s i o n o f g e n e s i n v o l v e d i n s e r i n e a n d o n e -

c a r b o n m e t a b ol i s m , i n c l u d i n g P h g d h , P s a t 1 , S h m t 2 ,

a n d A l d h 1 l 2 ( F i g u r e s 8 D t o 8 I ) . T h e s e r e s u l t s s u g g e s t

t h a t C n Ab 1 i s n e c e s s a r y f o r n o r m a l e n e r g y h o m e o -

s t a s i s i n t h e h e a r t a n d t h a t t h e d i s t u r b a n c e o f t h i s

p a t h w a y e x a c e r b a t e s c a r d i a c h y p e r t r o p h y .

D I S C U S S I O N

C a l c i n e u r i n a c t i v a t i o n p l a y s a c e n t r a l r o l e i n t h e

d e v e l o p m e n t o f m a l a d a p t i v e c a r d i a c h y p e r t r o p h y .

I n c o n t r a s t w i t h t h i s v i e w , w e s h o w h e r e t h a t

o v e r e x p r e s s i o n o f t h e a l t e r n a t i v e s p l i c i n g C n Ab

v a r i a n t C n Ab 1 r e d u c e s c a r d i a c h y p e r t r o p h y a n d i m -

p r o v e s e n e r g y p r o d u c t i o n , w h e r e a s d e l e t i o n o f i t s

C - t e r m i n a l u n i q u e d o m a i n r e s u l t s i n s p o n t a n e o u s

c a r d i a c h y p e r t r o p h y a n d d e c r e a s e d A T P s y n t h e s i s ,

s u g g e s t i n g t h a t t h e m e t a b o l i c e f f e c t s o f c a l c i n e u r i n

o n t h e h e a r t m a y b e i s o f o r m - s p e c i fic .

I n t e r e s t i n g l y , w e f o u n d t h a t C n Ab 1 o v e r e x p r e s s i o n

a c t i v a t e s s e r i n e a n d o n e - c a r b o n m e t a b o l i s m i n a n

m T O R - d e p e n d e n t m a n n e r . T h i s p a t h w a y i s a m a j o r

s o u r c e o f N A D H a n d N A D P H , a n d s u p p l i e s m e t a b o -

l i t e s t o m a j o r m e t a b o l i c p a t h w a y s i n t h e c e l l s ,

i n c l u d i n g n u c l e o t i d e b i o s y n t h e s i s , a m i n o a c i d h o -

m e o s t a s i s , m e t h y l a t i o n r e a c t i o n s , a n d a n t i o xi d a n t

d e f e n s e ( 1 2 , 1 9 ) . T h e s e r i n e a n d o n e - c a r b o n p a t h w a y

h a s r e c e n t l y g a i n e d s t r o n g a t t e n t i o n i n t h e c a n c e r

C E N T R A L I L L U S T R A T I O N b

Padrón-Barthe, L. et al. J Am Coll Cardio l. 2018;71(6):654 67.–

CnAb 1 activates mTOR and ATF4 to induce serine and one-carbon metabolism and promote the production of GSH, among other mediators.

Increased GSH results in reduced mitochondrial protein oxidation and increased ATP production, which improves cardiac function and

remodeling after pressure overload. ATF4 activating transcription factor 4; ATP adenosine triphosphate; G¼ ¼ b L G protein beta subunit-like;¼

GSH reduced glutathione; mTOR mechanistic target of rapamycin.¼ ¼



665

r e s e a r c h fie l d ( 1 2 ) . M a n y t u m o r c e l l s a r e d e p e n d e n t

o n s e r i n e a n d i n c r e a s e P h g d h a n d P s a t 1 e x p r e s s i o n t o

i n c r e a s e s e r i n e a v a i l a b i l i t y , w h i c h p r o v i d e s t h e s e

c e l l s w i t h a m e t a b o l i c a d v a n t a g e ( 2 0 ) . S e r i n e a n d o n e -

c a r b o n m e t a b o l i s m i s a l s o t r i g g e r e d b y m i t o c h o n d r i a l

d y s f u n c t i o n ( 2 1 , 2 2 ) . I t s r o l e i n t h e h e a r t , h o w e v e r , i s

v i r t u a l l y u n k n o w n .

H e r e , w e sh o w f o r t h e fir s t t i m e t o o u r k n o w l e d g e

t h a t s e r i n e a n d o n e - c a r b o n m e t a b o l i sm h a s a b e n e -

fic i a l e f f e c t o n t h e h e a r t , n e c e s s a r y f or t h e i m p r o v e d

A T P s y n t h e s i s a n d c a r d i a c f u n c t i o n i n d u c e d b y

C n Ab 1 . A c t i v a t i o n o f t h i s p a t h w a y l e a d s t o t h e

g e n e r a t i o n o f a n t i o x i d a n t m e t a b o l i t e s t h a t m a y b e

p r o t e c t i v e a g a i n s t r e a c t i v e o x y g e n s p e c i e s , e s p e -

c i a l l y G S H . G S H p r o d u c t i o n i s r e d u c e d f o l l o w i n g

p r e s s u r e o v e r l o a d , a n d t h i s r e d u c t i o n h a s a c a u s a l

e f f e c t o n t h e d e ve l o p m e n t o f c a r d i a c h y p e r t r o p h y ,

fib r o s i s , a n d r e m o d e l i n g t h a t l e a d s t o a d e c l i n e i n

c a r d i a c f u n c t i o n a f t e r T A C ( 2 3 ) . Co n s i s t e n t w i t h th i s

b e n e fi c i a l e f f e c t o f G S H o n t h e h y p e r t r o p h i c h e a r t ,

w e o b s e r ve d t h a t i n h i b i t i o n o f g l u t a t h i o n e s y n t h e s i s

p r e v e n t s t h e f u n c t i o n a l i m p r o v e m e n t i n d u c e d b y

C n Ab1 .

W e f o u n d t h a t N F A T i s e q u a l l y a c t i v a t e d i n W T a n d

aM H C - C n Ab 1 m i c e a f t e r T A C , s u g g e s t i n g t h a t C n Ab1 i s

n o t a c t i n g a s a n i n d i r e c t i n h i b i t o r o f N F A T . I n s t e a d ,

C n Ab 1 a c t i v a t e s t h e m e c h a n i s t i c t a r g e t o f r a p a m y c i n

( m T O R ) / A k t p a t h w a y a n d t h e t r a n s c r i p t io n f a c t o r

A T F 4 . W e h a v e p r e v i o u s l y r e p o r t e d t h a t C n Ab1 a c t i-

v a t e s t h i s p a t h w a y b y d i r e c t i nt e r a c t i o n o f i t s C - t e r -

m i n a l d o m a i n w i t h t h e m T O R C2 c o m p l e x a n d t h a t i t

i n d u c e s A T F 4 i n a n m T O R - d e p e n d e n t m a n n e r ( 4 , 6 ) .

S e r i n e a n d o n e - c a r b o n g e n e s a r e k n o w n t o b e r e g u -

l a t e d b y A T F 4 u p o n a c t i v a t i o n o f m T O R ( 1 0 , 1 1 , 2 1 ) .

T h e s e d a t a , t o g e t h e r w i t h t h e i n h i b i t i o n o f t h e s e

g e n e s b y r a p a m y c i n i n o u r t r a n s g e n i c m i c e , s u g g e s t

t h a t a c t i v a t i o n o f t h e m T O R / A T F 4 p a t h w a y b y C n Ab1

i n d u c e s m e t a b o l i c c h a n g e s t h a t a l l o w t h e c a r -

d i o m y o c y t e t o o b t a i n e n e r g y a n d N A D H / N A D P H

t h r o u g h t h e s e r i n e a n d o n e - c a r b on p a t h w a y ,

d e c r e a s i n g m i t o c h o n d r i a l p r o t e i n o x i d a t i on a n d p r e -

s e r v i n g A T P p r o d u c t i o n a f t e r p r e s s u r e o v e r l o a d

(C e n t r a l Il l u s t r a t i o n ) .

W e a l s o r e p o r t h e r e t h e fi r s t d e s c r i p t i o n o f m i c e

l a c k i n g C n Ab 1 f o l l o w i n g t h e d e l e t i o n o f C n Ab i n t r o n

1 2 - 1 3 . C n AbDi 1 2 m i c e a r e v i a b l e a n d f e r t i l e , a n d s h o w

n o o b v i o u s m a l f o r m a t i o n d u r i n g e m b r y o n i c d e v e l -

o p m e n t . H o w e v e r , t h e y d e v e l o p c a r d i a c h y p e r t r o p h y

a t 1 5 m o n t h s o f a g e . T h i s i s a c c o m p an i e d b y a

r e d u c t i o n i n A T P p r o d u c t i o n a n d a d e c l i n e i n c a r d i a c

f u n c t i o n . I n a d d i t i o n , w e s h o w t h a t C n AbDi 1 2 m i c e

d e v e l o p i n c r e a s e d c a r d i a c h y p e r t r o p h y a f t e r T A C . W e

o b s e r v e d a n i m p r o v e m e n t i n L V E F i n t h e s e m i c e ,

w h i c h w a s l i k e l y t h e d i r e c t r e s u l t o f h y p e r t r o p h y i t -

s e l f , b e c a u s e c a r d i a c f u n c t i o n h a d n o t y e t d e c o m -

p e n s a t e d i n t h e s e a n i m a l s b y 2 1 d a y s . C nAbD i 1 2 m i c e

s h o w e d d e c r e a s e d e x p r e s s i o n o f e n z y m e s i n v o l v e d i n

t h e s e r i n e s y n t h e s i s p a t h w a y , a f t e r e i t h e r T A C o r

s h a m s u r g e r y , s u g g e s t i n g t h a t a c t i v a t i o n o f t h i s

p a t h w a y by C n A b 1 i s n e c e s s a r y f o r t h e m a i n t e n a n c e

o f e n e r g y b a l a n c e a n d c a r d i a c h o m e o s t a s i s .

S T U D Y L I M I T A T I O N S . T h i s s t u d y w a s p e r f o r m e d i n

m i c e . A l t h o u g h w e s h o w e v i d e n c e o f t h e b e n e fic i a l

e f f e c t s o f C n Ab 1 o v e r e x p r e s s i o n a n d a c t i v a t i o n o f t h e

s e r i n e a n d o n e - c a r b o n p a t h w a y i n t h e m o u s e h e a r t ,

t r a n s l a t i o n o f r e s u l t s o b t a i n e d i n m i c e t o h u m a n s i s

n o t a l w a y s s t r a i g h t f o r w a r d .

C O N C L U S I O N S

O u r r e s u l t s d e m o n s t r a t e t h a t C n Ab 1 h a s a n o p po s i t e

e f f e c t o n t h e h e a r t t o t h a t o f o t h e r c a l c i n e u r i n i s o -

f o r m s . W e u n v e i l t h e a c t i v a t i o n o f t h e s e r i n e a n d o n e -

c a r b o n m e t a b o l i c p a t h w a y b y C n Ab1 a n d s h o w i t s

b e n e fic i a l r o l e i n t h e h e a r t f o r t h e fir s t t i m e . T h e s e

c h a n g e s i n o u r u n d e r s t a n d i n g o f c e l l s i g n a l i n g a n d

m e t a b o l i s m d u r i n g p r e s s u r e o v e r l o a d h y p e r t r o p h y

m a y l e a d t o t h e d e v e l o p m e n t o f n o v e l t h e r a p e u t i c

a v e n u e s b a s e d o n t h e m o d u l a t i o n o f s e r i n e a n d o n e -

c a r b o n m e t a b o l i s m .

A D D R E S S F O R C O R R E S P O N D E N C E : D r . E n r i q u e

L a r a - P e z z i , M y o c a r d i a l P a t h o p h y s i o l o g y A r e a , C e n t r o

N a c i o n a l d e I n v e s t i g a c i o n e s C a r d i o v a s c u l a r e s

C a r l o s I I I ( C N I C ) , M e l c h o r F e r n á nd e z A l m a g r o 3 ,

2 8 0 2 9 M a d r i d , S p a i n . E - m a i l : e l a r a @ c n i c . e s .

P E R S P E C T I V E S

COMPETENCY IN MEDICAL KNOWLEDGE:

CnAb 1 overexpression in cardiomyocytes reduces left

ventricular hypertrophy and remodeling , and im-

proves systolic function following pressure overload.

CnAb 1 promotes metabolic reprogramming of the

myocardium, activating the serine one-carbon

pathway, and generation of several antioxidant me-

tabolites. These metabolic changes are necessary to

preserve ATP synthesis and improve cardiac function

in CnAb 1-overexpressing mice.

TRANSLATIONAL OUTLOOK: Further research is

needed to explore the therapeutic potential of serine

one-carbon pathway activation for patients with heart

failure.



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K E Y W O R D S

A P P E N D I X



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