aerobic training induces lvh involving micrornas - fapesp · aerobic training induces lvh involving...
TRANSCRIPT
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
No cardiac dysfunction
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
No cardiac dysfunction
Pressure overload
Without chamber dilation
Myocyte width >> Myocyte length
No fibrosis
No cardiac dysfunction
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
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
Humano
21
7
Constrição aórtica
Knock in Caucineurina A
Van Rooij E. et al. Proc Natl Acad Sci U S A. 2006 .
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;
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.
Moderate-volume training Moderate Cardiac Hypertrophy
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.
Moderate-volume training Moderate Cardiac Hypertrophy
High-volume training Robust Cardiac Hypertrophy
High performance, like an atleta
Oliveira et al., JRAAS, 2009.
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
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
p<0.05 vs SC
Pathological Cardiac Hypertrophy Markers
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
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
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.
Cardiac ACE-2 - Ang (1-7) axis
Fernandes, et al., Hypertension, 58: 182-189, 2011
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
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.
- 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
Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)
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
Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)
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
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
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
)
Silva-Jr ND et al., Medicine & Science in Sports & Exercise (em revisão)
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
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 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
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
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%
*
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
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
*
*
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
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
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
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
Soci, UPR et al., Physiological Genomics 43: 665-673, 2011
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