aerobic training induces lvh involving micrornas - fapesp · aerobic training induces lvh involving...

Aerobic training induces LVH involving

microRNAs

Edilamar Menezes de Oliveira [email protected]

Grant FAPESP (2009/18370-3)

Grant CNPq/MCT-2009

Laboratory of Biochemistry and Molecular Biology of the Exercise

School of Physical Education and Sport

University of Sao Paulo - Brazil

Morfológicas/Funcionais

Diretriz de Reabilitação Cardíaca, SBC, 2005 Oliveira & Krieger, 2002

Rno-mir-1

Hemodinâmicas

PA de repouso Fluxo Sanguíneo Volume plasmático

Cardiovasculares

FC repouso Melhor Função Ventricular (VS) Angiogênese

HIPERTROFIA

CARDÍACA FISIOLÓGICA

Metabólicas VO2max Estrutura miofibrilar Síntese e atividade enzimática

Adaptações Fisiológicas ao Treinamento Físico Aeróbio

Fernandes T et al. BJMBR, Sep, 2011

Nadal-Ginard & Madavi, J Clin Invest, 1989.

Heineke & Molkentin Nat Rev, 2006.

Physiological Cardiac Hypertrophy

RV LVRV LVLVRV

SEDENTARY

Eccentric Hypertrophy

AEROBIC TRAINING RESISTANCE TRAINING

Concentric HypertrophyVolume overload

Chamber dilation

Myocyte length >> Myocyte width

No fibrosis

No cardiac dysfunction

Pressure overload

Without chamber dilation

Myocyte width >> Myocyte length

No fibrosis


sarcomeres

RV LVRV LVRV LVRV LVLVLVRV LVRV

SEDENTARY

Eccentric Hypertrophy

AEROBIC TRAINING RESISTANCE TRAINING

Concentric HypertrophyVolume overload

Chamber dilation

Myocyte length >> Myocyte width

No fibrosis


Pressure overload

Without chamber dilation

Myocyte width >> Myocyte length

No fibrosis


sarcomeres

DORN, Hypertension , 2007.

INFLAMA Ç Ã O

APOPTOSE

GLIC Ó LISE

GENES FETAIS

S Í NTESE DE

PROTE Í NA

MATRIX

EXTRACELULAR

BIOG Ê NESE

MITOCONDRIA

METABOLISMO

OXIDATIVO

CAPILARIZA ÇÃ O

HIPERTROFIA DE

CARDIOMI Ó CITOS

Some miRNAs are specifically expressed in specific

tissues, such as heart

MicroRNAs are short sequences

non coding protein

regulate specific target genes by

inhibiting translation

MicroRNAs

Descoberta

Lee RC, Feinbaum RL, Ambros V. Cell,1993.

Victor Ambros

Rosalind Lee

Descoberta

Reinhart, B. et al. Nature , 2000.

Lin-14

Lin-28

Lin-41

Lin-42

Daf-12

Descoberta em Humano

Pasquinelli, AE. et al. Nature , 2000.

2002

– Correlation between miRNAs e Câncer

2005

– Dicer deletion indicating miRNA relevance in

diferent model systems

- Antagomir inhibition of mRNAs in vivo

- First miRNA transgênic mouse

2006 - Correlation between miRNAs and CV disease

MicroRNAs e HC

Van Rooij E. et al. Proc Natl Acad Sci U S A. 2006 .

Eva van Rooij

Eric Olson

Humano

21

7

Constrição aórtica

Knock in Caucineurina A

Van Rooij E. et al. Proc Natl Acad Sci U S A. 2006 .

Care A. et al. Nature Medicine, 2007.

Gianluigi Condorelli

Van Rooij E. et al. Science, 2007.

O Exercício Físico poderia influenciar na expressão de microRNAs no remodelamento

cardíaco?

We investigate the role of microRNAs

regulating LV hypertrophy induced by aerobic

training in normotensive rats:

- Classic and the novel cardiac renin angiotensin

system (RAS);

- Angiogenesis;

- Collagen.

Training Protocols

Swimming training 1 (T1): 60 min/day;

1x day;

5x/week;

during 10 weeks


1x/ day;

5x/week;

during 8 weeks

9a week: 60 min; 5x/week; 2x/ day


Oliveira et al., JRAAS, 2009.

Training Protocols


1x day;

5x/week;

during 10 weeks


1x/ day;

5x/week;

during 8 weeks




Moderate-volume training Moderate Cardiac Hypertrophy

Training Protocols


1x day;

5x/week;

during 10 weeks


1x/ day;

5x/week;

during 8 weeks




Moderate-volume training Moderate Cardiac Hypertrophy

High-volume training Robust Cardiac Hypertrophy

High performance, like an atleta


Swimming training System

Workload: caudal dumbbells weighting 5% of corresponding body

weight

Exercise training markers

* p<0.05, vs SC. Soci, UPR et al., Physiological Genomics 43: 665-673, 2011

Fernandes, et al., Hypertension, 58: 182-189, 2011

Heart rate

SC T1 T2

*

*

0

150

300

450

600

S T1 T2

Citrate synthase activity

SC

T1

T2

* *

SC T1 T2

CS

act

ivit

y

(µm

ol/

ml/

mg

pro

tein

)

Cardiovascular Marker

Metabolic Marker

A CB C T1 T2

0

30

60

90

120

150

ECO (mg/g) LV/BW (mg/g) Myocyte Diameter (µm)

Card

iac

Hyp

ert

rop

hy

(% o

f c

on

tro

l) *

* * **

** *

† S T1 T2

S T1 T2

Cardiac Hypertrophy

Soci, UPR et al., Physiological Genomics 43: 665-673, 2011

y = 17,26x + 25,48

R = 0.680018

P<0.05

55

65

75

85

95

2.3 2.5 2.7 2.9 3.1 3.3 3.5

VO

2m

ax

(m

l.k

g-1

.min

-1 )

Cardiac Hypertrophy

(mg/g)

Positive Correlation

VO2max X Cardiac Hypertrophy


p<0.05 vs SC

Pathological Cardiac Hypertrophy Markers


microRNA Array (349 miRNAs)

87 microRNAs were

differentially expressed with

exercise training

- 10.000 20.000 30.000 40.000 50.000 60.000

miRNA

- 2.000 4.000 6.000 8.000 10.000

miRNA

P2 P1 SC

* p<0.01 vs SC.

T2

T1

SC

miRNAs differentially expressed in the

LV with Exercise training.

Increased by T1

SC

Decreased by T1

Largest decreased by T2

Largest increased by T2

miRs, 1, 133a e 133b Expression

* p<0.01 vs SC. Soci, UPR et al., Physiological Genomics 43: 665-673, 2011

0

10000

20000

30000

40000

50000

60000

70000

1

Un

ida

de

s a

rbit

rári

as

de

ex

pre

ss

ão

gê

nic

a

SC

P1

P2

miR-133a miR-133b miR-1

m

iRN

As L

evels

(U

.A.)

T1

T2





system (RAS);

- Angiogenesis;

- Collagen.


Cardiac ACE - Ang II axis

Cardiac ACE-2 - Ang (1-7) axis


Cardiac ACE-2 - Ang (1-7) axis


These results suggest that this nonclassic cardiac RAS

counteracts the classic RAS

This modulation might increase blood and oxygen transport to the

exercising cardiac muscle to facilitate high performance

miR-143

VASODILATION

VASOCONSTRICTION

ACE-2

CH

miR-27a

miR-27b

ACE

microRNA and

Physiological Hypertrophy

Ang (1-7)

Ang II





system (RAS);

- Angiogenesis;

- Collagen.

- 5.000 10.000 15.000 20.000 25.000

rno-let-7f

rno-miR-126

rno-let-7b

rno-miR-221

rno-miR-222

rno-miR-27b

rno-miR-378

rno-miR-214

Expressão gênica (Unidades Arbitrárias)

SC

P1

P2

microRNAs Angiogênicos

Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)

Representação esquemática da ação do miR-126 em vias angiogênicas mediadas por VEGF


0

50

100

150

200

250

SC P1 P2

*

*

†

SC

P1

P2

Exp

ress

ão r

elati

va d

o V

EG

F n

o V

E

por

wes

tern

blo

ttin

g(%

do c

on

trole

)

microRNA e Angiogênese Cardíaca


microRNA-126 no VE

0

20

40

60

80

100

120

140

160

180

200

SC P1 P2

Exp

ressão r

ela

tiva d

o m

iR-1

26 n

o V

Ep

or m

icro

arra

y(%

do

co

ntr

ole

) ******† † †

A

0

20

40

60

80

100

120

140

160

SC P1 P2

po

r r

ea

l-ti

me P

CR

(%

do

co

ntr

ole

)

Ex

pressã

o r

ela

tiv

a d

o m

iR-1

26

no

VE

***

SC

P1

P2

B

0,4

0,6

0,8

1,0

1,2

1,4

1,6

0,0 0,5 1,0 1,5 2,0 2,5m

iR-1

26 (

2 -

Ct )

Razão capilar / fibra no VE(no capilares / fibra)

R = 0,63

P < 0,05


Representação esquemática da ação do miR-126 em vias angiogênicas mediadas por VEGF

0

20

40

60

80

100

120

SC P1 P2

Exp

ress

ão r

ela

tiva d

o P

I3K

R2 n

o V

E

por r

eal

tim

e –

PC

R (

% d

o c

on

trole

)

SC

P1

P2

*

*** †

0

20

40

60

80

100

120

SC P1 P2

SC

P1

P2

* *E

xp

ress

ão

rela

tiv

a d

o S

pred

-1 n

o V

E

po

r w

est

ern

blo

ttin

g(%

do

co

ntr

ole

)


miR-126

VEGF

VEGFR2

PI3K

Akt

eNOS

Raf-1

MEK 1/2

PI3KR2 Spred-1

miR-126

ERK 1/2

Treinamento

de Natação

ANGIOGÊNESE CARDÍACA





system (RAS);

- Angiogenesis;

- Collagen.


miR-29 Family

MicroRNAs 29a, b e c:

Cardiac Fibrosis Target Genes

Van Rooij et al, PNAS, 2008

Target Genes Validated

COL1 A1

COL1 A2

COL3 A1

ELN

FBN1

MicroRNA 29

Collagen Fibrilin Elastin

miRNA-1 miRNA-133a miRNA-133b miRNA-29c 0

50

100

150

200

250

300

* * * * * *

*

* S

T1

T2

S

T1

T2

Rela

tive

LV

miR

NA

s l

eve

ls

by r

ea

l-ti

me

PC

R (

% o

f c

on

tro

l)

miRNAs Expression by Real Time-PCR

* p<0.05, vs. S


Collagen Expression

* p<0.05 vs. S

0

25

50

75

100

125

COLIAI COLIIIAI

Re

lati

ve E

xpre

ssio

n o

f LV

Co

llage

n(%

of

con

tro

l)

S

T1

T2

*

**

40% 49% 61% 52%

*


Cardiac Collagen Concentration and miRNA-29

0

70

140

210

280

S T1 T2

LV

mic

roR

NA

-29c le

vels

(%

of

co

ntr

ol)

MiRNA29c

*

*


0

30

60

90

120

150

S T1 T2

Car

dia

c O

H-P

rolin

(m

g/g)

(% o

f c

on

tro

l) * *

Colágeno cardíaco Cardiac Collagen

miRNA-29 Expression was inversaly correlated to

OH-Proline concentration in the heart

MiRNA- 29c (2 -ΔΔCT)

0

100

200

300

1 2 3

OH

-pro

lin

e (

mg

/g)

R = -0.61

P<0.05

0


The collagen concentration decreased

was associated with improvement of

ventricular compliance and function

Physiological Genomics 43: 665-673, 2011

SC (n=7)

T1 (n=6)

T2 (n=6)

Systolic Function

EFj (%)

77±5

75±2

73±2

EFn (%)

39±4

38±3

36±2

VEC (circ/s)

0.005±0.02

0.004±0.2

0.004±0.04

Diastolic Fuction

Peak E (m/s)

0.451±0.05

0.499±0.05

0.458±0.03

Peak A (m/s)

0.328±0.06

0.333±0.03

0.279±0.01

Ratio E/A

1.396±0.16

1.504±0.19

1.644±0.11*

IVRT(ms)

30.2±1.7

27.0±1.8*

* p<0.05, vs. S

Echocardiography

30.7±1.9

Global Function

DTPE (ms)

2.2±0.2 1.8±0.1* 1.9±0.2*

MPI

0.51±0.06 0.35±0.09* 0.38±0.07*

IVRT: isovolumetric relaxation time;

DTPE: deceleration time of peak E;

MPI: Myocardial Performance Index


Summary

ET

Diastolic Function

COLI and COLIII Expression Cardiac COL Concentration Pathological Cardiac Markers

miR-29c

Physiological

Cardiac Hypertrophy

Fernandes T et al. BJMBR, 2011.

AEROBIC TRAINING

miR-1, 133a and 133b miR-29a, 29b and 29c miR-27a and 27b miR-143

Rhoa/ CDC42

NELFA/ Whsc2

COLIAI

COLIIIAI

ACE ACE2

LV

PHYSIOLOGICAL

CARDIAC

HYPERTROPHY

Differentiation

and growth cell

Ventricle

Compliance

Vasodilation

Anti-fibrosis

Vasodilation

Anti-fibrosis

AEROBIC TRAINING

miR-1, 133a and 133b miR-29a, 29b and 29c miR-27a and 27b miR-143

Rhoa/ CDC42

NELFA/ Whsc2

COLIAI

COLIIIAI

ACE ACE2

LVLV

PHYSIOLOGICAL

CARDIAC

HYPERTROPHY

Differentiation

and growth cell

Ventricle

Compliance

Vasodilation

Anti-fibrosis

Vasodilation

Anti-fibrosis

Together these effects induce regulation of

cardioprotector genes, improve ventricular

compliance, and might provide the additional aerobic

capacity required by the exercised heart.

These results suggests that a basis for treatment to

prevent of the development of pathological LVH

might be to inhibit specific miRNAs, using antisense

or siRNA.

Perspectivas Futuras

Estudos com Animais Experimentais:

-SHR - Bolsa de Mestrado (FAPESP (Projeto 2009/03264-3)

-Insuficiência cardíaca – Bolsa de Doutorado (FAPESP (Projeto 2010/09438-0)

Estudo com Humanos: (Colaboração com Prof. Carlos Eduardo Negrão e Profa.

Maria Urbana Rondon).

-Pacientes Hipertensos

-Pacientes Resincronizados

Acknowledgments Pos-graduation Students

Tiago Fernandes

Ursula Soci

Marco Amadeu

Nara Hashimoto

Flávio Magalhães

Kaleizu Rosa -InCor

Colaborations:

Prof. Maria Cláudia Irigoyen and José Eduardo Krieger – InCor

Prof. Adriana Carmona and Dulce Casarini - UNIFESP

Dr. M Ian Phillips – KGI –California – USA.

Laboratory Technician

Glória Motta

Laboratory of Biochemistry and Molecular Biology of the Exercise

Thank you for your attention

aerobic training induces lvh involving micrornas - fapesp · aerobic training induces lvh involving...

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