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
Adaptações Fisiológicas ao
Treinamento Físico Aeróbio
Hemodinâmicas
PA de repouso
Fluxo Sanguíneo
Volume plasmático
Cardiovasculares
FC repouso
Rno-mir-1
Morfológicas/Funcionais
Metabólicas
VO2max
Estrutura miofibrilar
Síntese e atividade
enzimática
Melhor Função
Ventricular (VS)
Angiogênese
HIPERTROFIA
CARDÍACA
FISIOLÓGICA
Diretriz de Reabilitação Cardíaca, SBC, 2005
Oliveira & Krieger, 2002
Physiological Cardiac Hypertrophy
AEROBIC TRAINING
RV
LV
SEDENTARY
RV
Eccentric Hypertrophy
Volume overload
Chamber dilation
Myocyte length >> Myocyte width
No fibrosis
No cardiac dysfunction
Fernandes T et al. BJMBR, Sep, 2011
Heineke & Molkentin Nat Rev, 2006.
Nadal-Ginard & Madavi, J Clin Invest, 1989.
LV
RESISTANCE TRAINING
RV
LV
sarcomeres
Concentric Hypertrophy
Pressure overload
Without chamber dilation
Myocyte width >> Myocyte length
No fibrosis
No cardiac dysfunction
HIPERTROFIA DE
CARDIOMIÓCITOS
METABOLISMO
OXIDATIVO
BIOGÊNESE
MITOCONDRIA
CAPILARIZAÇÃO
APOPTOSE
INFLAMAÇÃO
SÍNTESE DE
PROTEÍNA
GLICÓLISE
MATRIX
EXTRACELULAR
GENES FETAIS
DORN, Hypertension, 2007.
MicroRNAs
MicroRNAs are short sequences
non coding protein
regulate specific target genes by
inhibiting translation
Some miRNAs are specifically expressed in specific
tissues, such as heart
Descoberta
Victor Ambros
Rosalind Lee
Lee RC, Feinbaum RL, Ambros V. Cell,1993.
Descoberta
Lin-14
Lin-28
Lin-41
Lin-42
Daf-12
Reinhart, B. et al. Nature , 2000.
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
Eva van Rooij
Eric Olson
Van Rooij E. et al. Proc Natl Acad Sci U S A. 2006 .
Constrição aórtica
Humano
Knock in
Caucineurina A
21
7
Van Rooij E. et al. Proc Natl Acad Sci U S A. 2006
Gianluigi Condorelli
Care A. et al. Nature Medicine, 2007.
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
Swimming training 2 (T2): 60 min/day;
1x/ day;
5x/week;
during 8 weeks
9a week: 60 min; 5x/week; 2x/ day
10a week: 60 min; 5x/week; 3x/ day
Oliveira et al., JRAAS, 2009.
Training Protocols
Swimming training 1 (T1): 60 min/day;
1x day;
Moderate-volume training
Moderate Cardiac Hypertrophy
5x/week;
during 10 weeks
Swimming training 2 (T2): 60 min/day;
1x/ day;
5x/week;
during 8 weeks
9a week: 60 min; 5x/week; 2x/ day
10a week: 60 min; 5x/week; 3x/ day
Oliveira et al., JRAAS, 2009.
Training Protocols
Swimming training 1 (T1): 60 min/day;
1x day;
Moderate-volume training
Moderate Cardiac Hypertrophy
5x/week;
during 10 weeks
Swimming training 2 (T2): 60 min/day;
1x/ day;
High-volume training
Robust Cardiac Hypertrophy
5x/week;
High performance, like an atleta
during 8 weeks
9a week: 60 min; 5x/week; 2x/ day
10a week: 60 min; 5x/week; 3x/ day
Oliveira et al., JRAAS, 2009.
Swimming training System
Workload: caudal dumbbells weighting 5% of corresponding body
weight
Oliveira et al., JRAAS, 2009.
Exercise training markers
Heart rate
Cardiovascular Marker
CS activity
(µmol/ml/mg protein)
SC
600
T2
T1
Citrate synthase activity
450
* *
300
* *
SC
T1
T2
150
* p<0.05, vs SC.
Metabolic Marker
0
SC
S
T1
T1
T2
T2
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
Fernandes, et al., Hypertension, 58: 182-189, 2011
Cardiac Hypertrophy
†
Cardiac Hypertrophy
(% of control)
150
120
*
**
*
**
*
*
S
T1
T2
90
60
30
0
ECO (mg/g)
A
C
LV/BW (mg/g)
Myocyte
Diameter (µm)
B
T1
C
T2
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
Positive Correlation
VO2max X Cardiac Hypertrophy
VO2max (ml.kg-1.min-1 )
95
85
75
y = 17,26x + 25,48
65
R = 0.680018
P<0.05
55
2.3
2.5
2.7
2.9
3.1
3.3
3.5
Cardiac Hypertrophy
(mg/g)
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
Pathological Cardiac Hypertrophy Markers
p<0.05 vs SC
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
miRNA
miRNA
microRNA Array
T2
P2
T1
P1
SC
SC
(349 miRNAs)
87 microRNAs
were
differentially
expressed with
exercise training
* p<0.01 vs SC.
-
-
10.000
20.000
30.000
40.000
50.000
60.000
2.000
4.000
6.000
8.000
10.000
miRNAs differentially expressed in the
LV with Exercise training.
SC
Increased by T1
Largest increased by T2
Decreased by T1
Largest decreased by T2
(U.A.)
Levels de
miRNAs
expressão gênica
arbitrárias
Unidades
miRs, 1, 133a e 133b Expression
70000
60000
SC
50000
P1
T1
40000
P2
T2
30000
20000
10000
0
miR-1
* p<0.01 vs SC.
1
miR-133a
miR-133b
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
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.
Fernandes, et al., Hypertension, 58: 182-189, 2011
Fernandes, et al., Hypertension, 58: 182-189, 2011
Cardiac ACE - Ang II axis
Fernandes, et al., Hypertension, 58: 182-189, 2011
Cardiac ACE-2 - Ang (1-7) axis
Fernandes, et al., Hypertension, 58: 182-189, 2011
Cardiac ACE-2 - Ang (1-7) axis
These results suggest that this nonclassic cardiac RAS
counteracts the classic RAS
Fernandes, et al., Hypertension, 58: 182-189, 2011
Fernandes, et al., Hypertension, 58: 182-189, 2011
microRNA and
Physiological Hypertrophy
miR-143
miR-27a
miR-27b
ACE-2
ACE
VASODILATION
VASOCONSTRICTION
Ang (1-7)
CH
Ang II
This modulation might increase blood and oxygen transport to the
exercising cardiac muscle to facilitate high performance
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.
microRNAs Angiogênicos
rno-miR-214
rno-miR-378
rno-miR-27b
SC
P1
P2
rno-miR-222
rno-miR-221
rno-let-7b
rno-miR-126
rno-let-7f
-
5.000
10.000
15.000
20.000
25.000
Expressão gênica (Unidades Arbitrárias)
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
Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)
Expressão relativa do VEGF no VE
por western blotting (% do controle)
microRNA e Angiogênese Cardíaca
†
250
*
200
SC
P1
*
P2
150
100
50
0
SC
P1
P2
Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)
microRNA-126 no VE
**
160
*
140
1,6
SC
100
P1
P2
80
60
Ct )
1,4
120
miR-126 (2 -
Expressão relativa do miR-126 no VE
por real-time PCR (% do controle)
B
1,2
1,0
R = 0,63
0,8
P < 0,05
0,6
40
0,4
20
0,0
0
SC
P1
P2
0,5
1,0
1,5
2,0
2,5
Razão capilar / fibra no VE
(no capilares / fibra)
Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)
120
120
100
*
80
SC
P1
60
P2
†
40
***
20
0
SC
P1
P2
Expressão relativa do Spred-1 no VE
por western blotting (% do controle)
Expressão relativa do PI3KR2 no VE
por real time – PCR (% do controle)
Representação esquemática da ação do miR-126 em vias
angiogênicas mediadas por VEGF
100
80
*
*
SC
P1
60
P2
40
20
0
SC
P1
P2
Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)
VEGF
Treinamento
de Natação
VEGFR2
miR-126
PI3KR2
miR-126
PI3K
Raf-1
Akt
MEK 1/2
eNOS
ERK 1/2
ANGIOGÊNESE CARDÍACA
Spred -1
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.
miR-29 Family
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
MicroRNAs 29a, b e c:
Cardiac Fibrosis Target Genes
Target Genes
Validated
MicroRNA 29
COL1 A1
COL1 A2
COL3 A1
ELN
Collagen
Fibrilin
Elastin
FBN1
Van Rooij et al, PNAS, 2008
miRNAs Expression by
Real Time-PCR
Relative LV miRNAs levels
by real-time PCR (% of control)
300
250
S
T1
T2
*
200
*
150
100
50
* *
* *
*
*
0
miRNA-1
miRNA-133a
miRNA-133b
miRNA-29c
* p<0.05, vs. S
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
Collagen Expression
Relative Expression of LV Collagen
(% of control)
125
S
T1
100
T2
75
*
50
25
*
*
49%
40%
*
61%
52%
0
COLIAI
COLIIIAI
* p<0.05 vs. S
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
Cardiac Collagen Concentration and miRNA-29
MiRNA29c
LV microRNA-29c levels
(% of control)
280
*
210
*
140
70
0
S
150
T1
T2
Colágeno
cardíaco
Cardiac Collagen
Cardiac OH-Prolin (mg/g)
(% of control)
120
*
90
*
60
30
0
S
T1
T2
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
miRNA-29 Expression was inversaly correlated to
OH-Proline concentration in the heart
300
OH-proline (mg/g)
R = -0.61
P<0.05
200
100
0
0
1
MiRNA- 29c (2
2
3
-ΔΔCT)
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
The collagen concentration decreased
was associated with improvement of
ventricular compliance and function
Echocardiography
Systolic Function
EFj (%)
EFn (%)
VEC (circ/s)
SC (n=7)
T1 (n=6)
77±5
39±4
75±2
38±3
0.005±0.02
0.004±0.2
0.004±0.04
T2 (n=6)
73±2
36±2
Diastolic Fuction
Peak E (m/s)
Peak A (m/s)
0.451±0.05
0.499±0.05
0.458±0.03
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)
30.7±1.9
30.2±1.7
27.0±1.8*
DTPE (ms)
2.2±0.2
1.8±0.1*
1.9±0.2*
0.35±0.09*
0.38±0.07*
Global Function
MPI
0.51±0.06
IVRT: isovolumetric relaxation time;
DTPE: deceleration time of peak E;
MPI: Myocardial Performance Index
* p<0.05, vs. S
Physiological Genomics 43: 665-673, 2011
Summary
ET
miR-29c
COLI and COLIII Expression
Cardiac COL Concentration
Pathological Cardiac Markers
Physiological
Cardiac Hypertrophy
Diastolic Function
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
AEROBIC TRAINING
miR-1, 133a and 133b
Rhoa/ CDC42
NELFA/ Whsc2
Differentiation
and growth cell
miR-29a, 29b and 29c
miR-27a and 27b
miR-143
ACE
ACE2
COLIAI
COLIIIAI
Ventricle
Compliance
LV
Vasodilation
Anti-fibrosis
Vasodilation
Anti-fibrosis
PHYSIOLOGICAL
CARDIAC
HYPERTROPHY
Fernandes T et al. BJMBR, 2011.
 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
Laboratory Technician
Glória Motta
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 of Biochemistry and Molecular Biology of the Exercise
Thank you for your attention