Transcript Aging

MITOCHONDRIAL (DYS)FUNCTION IN THE AGED AND
FAILING MYOCARDIUM: SOME COMMON FEATURES
Marisol Ruiz-Meana
Vall d’Hebron Institut de Recerca, Barcelona (Spain)
Busan 8-10 December 2016
Human population is aging...
Number of people >65 years
Developed countries
Developing countries
Number of centenarians
New demographic model with
unknown medical, social and
economic impact
“The first person to live to 140 years old has already been born” (2016)
Dr. David Sinclair, biogerontologist. Harvard Medical School.
Aging and CV disease
Deaths per 100.000 population
Year
Percentage of population (%)
Prevalence of HF by age and sex
HF is an age-related condition
In Europe and US the estimated
prevalence of HF is 2%
Men
Women
The prevalence of HF is expected to
rise as a result of population aging
and the improved survival of CAD
20-39
40-59 60-79
Age (years)
80+
Lloyd-Jones et al. Circulation 2010
>300.000
Evolution of scientific publications on
AGING (PubMed)
Number of publications
14000
12000
10000
8000
6000
Aging AND heart
700
600
500
400
300
200
100
0
>20.000
2000 2002 2004 2006 2008 2010 2012
2015
4000
2000
0
2000 2002 2004 2006 2008 2010 2012
2015
>300.000
Evolution of scientific publications on
AGING (PubMed)
Number of publications
14000
12000
Aging AND heart
700
600
500
400
300
200
100
0
>20.000
10000J Mol Cell Cardiol 2015
So! What’s aging?
8000
6000
2000 2002 2004 2006 2008 2010 2012
2015
4000
2000
0
2000 2002 2004 2006 2008 2010 2012
2015
Aging phenotype: 7 types of damage
Mitochondrial dysfunction
DNA mutations
Cellular loss
Cell proliferation
Extracellular junk
Intracellular junk
Protein crosslinks
Aging still largely represents a collection of intriguing yet unconnected data
Aubrey de Grey. Gerontol 2013
Aging phenotype: 7 types of damage
Mitochondrial dysfunction
DNA mutations
ATP / calcium / ROS
Cellular loss
Cell proliferation
Extracellular junk
Intracellular junk
Protein crosslinks
Fibrosis
From aging to failing cardiomyocytes...
Aging
cardiomyocytes
Altered calcium handling
Bioenergetic deficit
Failing
cardiomyocytes
Excessive ROS production
Decreased antioxidant capacity
Reduced tolerance to stress
...
health
disease
Is there a common contribution of
mitochondria to aging and HF?
The exchange of calcium between SR and mitochondria
couples energy supply with energy demand
ROS
Oxidative
Efficient coupling in energy demand/supply
and
stress
OxPhos
ROS detox require close proximity between SR and
ATP
mitochondria
GSSG
NADPH
Contraction
Krebs
GSH
NADP+
Antioxidant
regeneration
Mitochondria
Myofilaments
Ca2+
RyR
SERCA
SR
...and regulates the regeneration of the antioxidant glutathione
Bioenergetic mismatch in aging cardiomyocytes
50,0
45,0
40,0
35,0
NAD(P)H regeneration under pacing
4-6 months
Young
Old
*
30,0
25,0
20,0
15,0
10,0
5,0
0,0
Resting
ADP-stimulated
ROS
90
80
70
60
50
40
30
20
10
0
50
Young
Old
45
40
35
30
5 Hz
25
0 20 40 60 80 100 120 140 160 180
ROS production (5 Hz pacing)
100
>20 months
NAD(P)H (% of total)
QO nmolsO/min.CS
Maximal O2 consumption
Time (s)
Young
Old
*
Cyto ROS
(DCF)
Mito ROS
(MitoSoX)
Mitochondria from aging cardiomyocytes fail
to adapt to energy demanding conditions
Fernandez-Sanz et al. Cell Death and Dis 2014
Interplay between SR and mitochondria
Young
Merge
Anti-RyR
Anti-VDAC
Old
20µm
Merge
Mander's coefficient (m1)
1.0
p<0.05
0.8
*
0.6
0.4
0.2
0.0
Young
Old
Aging reduces the anatomical
proximity between SR and
mitochondria
Anti-RyR
Anti-VDAC
Fernandez-Sanz et al. Cell Death and Dis 2014
In failing cardiomyocytes
DCF (ROS)
HF
Sham
MCB (Glutathione)
*
Sham
HF
Sham
*
Sham
HF
HF
Increased oxidative stress
Goh KY et al. Cardiovasc Res 2016
Disruption of mitochondria-SR network
Do mitochondria become “orphaned” in senescent
cardiomyocytes?
- Functional disruption from SR and/or
mitochondrial network
- Increased susceptibility to stress
Effect of aging on mitochondrial functional interplay
with SR
with other mitochondria
Defective calcium exchange with SR
1.06
Young
Old
Time to rigor (s)
Rhod-2 (a.u.)
1.08
Delayed propagation of mitochondrial
depolarization in ischemia
1.04
1.02
1.00
caffeine
0.98
0 15 20 25 30
35 40
45 50
Time (s)
*
500
450
400
350
300
250
200
150
100
50
0
Young
Old
In aging cells, mitochondria form defective functional microdomains (orphaned)
Aged (orphaned) mitochondria are less tolerant to stress
Decreased tolerance to calcium exposure
Young
Old
CG5N (a.u.)
6000
CsA
5000
4000
Ca 2+
3000
2000
1000
0
0
320 640 960 1280 1600 1920 2240 2560
Time (s)
Increased susceptibility to mPTP during I/R
young
old
(+ BDM)
0 min
3 min
5 min
Calcein release (a.u.)
1.0
0.9
0.8
0.7
0.6
Fernandez-Sanz et al. Thromb and Haemost 2016
0
2
4
6
8
10
12
Time (min)
Why do mitochondria become orphaned in the aging heart?
RyR-2
Mfn-2
SR
Grp-75
VDAC
Mito
At the microsomes:
Young Old
RyR
SERCA2
Grp75
(565 kDa)
Mfn2
VDAC1
(75 kDa)
GAPDH
(75 kDa)
(32 kDa)
(30 kDa)
a.u.
(110 kDa)
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Young
Old
Mfn2
VDAC1 Grp75 RyR2
In aging cardiomyocytes, disruption of SR-mitochondria is not due
to downregulation of Mfn-2, VDAC, RyR or Grp75
Chen Y. Circ Res 2012
How does aging modify RyR calcium release?
Effect of aging on RyR calcium kinetics
Spark frequency
7,0
5,0
1.00
4,0
3,0
2,0
1,0
0,0
*p<0.01
Young
Old
Fluo-4 AM (a.u.)
Sparks/100um/s
RyR-dependent calcium leak
*
6,0
0.95
0.90
Thapsigargin
0.85
0.80
0.75
0.70
0.65
Young
Old
Ru360
0
30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600
Time (s)
(Unpublished data)
SR from aging cardiomyocytes display altered RyR gating properties
and increased calcium leak
2+
2+
Ca2+Ca Ca2+Ca
Ca2+
mitochondria
Ca2+
Normal RyR-2
SR
Ca2+
Leaky RyR-2
From mice to men…
*
*
1000
800
600
400
* p<0.02
200
0
Young
Old
nmol Ca/CS
Rhod-2 (a.u.)
350
300
250
200
150
100
50
0
* p<0.01
<75 years ≥75 years
Calcium content is increased in mitochondria from aging hearts
(unpublished data)
Why does RyR become leaky in aging cells?
Oxidation
Phosphorylation
Nitrosation
Glycation
...
Advanced glycation end-products
(AGEs) accumulate in aging and
induce irreversible protein changes
Brownlee M. Annu Rev Med 1995/Metabolism 2000
Effect of aging on post-translational modification of RyR
Peptide sequence
EEKAKDEK
FAVFCNGESVEENANVVVR
FAVFCNGESVEENANVVVR
FAVFCNGESVEENANVVVR
FLPPPGYAACYEAVLPK
NVPPDLSICTFVLEQSLSVR
NVPPDLSICTFVLEQSLSVR
NVPPDLSICTFVLEQSLSVR
NVPPDLSICTFVLEQSLSVR
NVPPDLSICTFVLEQSLSVR
TDDVISCCLDLSAPSISFR
TDDVISCCLDLSAPSISFR
VLQDDEFTCDLFR
VAHALCSHVDEPQLLYAIENK
VAHALCSHVDEPQLLYAIENK
VAHALCSHVDEPQLLYAIENK
Type of modification (CML)
N-Term(TMT6plex); K5(HydroxymethylOP); K8(TMT6plex)
N-Term(TMT6plex); C5(Carboxymethyl)
N-Term(TMT6plex); C5(Carboxymethyl)
N-Term(TMT6plex); C5(Carboxymethyl)
N-Term(TMT6plex); C10(Carboxymethyl); K17(TMT6plex)
N-Term(TMT6plex); C9(Carboxymethyl)
N-Term(TMT6plex); C9(Carboxymethyl)
N-Term(TMT6plex); C9(Carboxymethyl)
N-Term(TMT6plex); C9(Carboxymethyl)
N-Term(TMT6plex); C9(Carboxymethyl)
N-Term(TMT6plex); C7(Carbamidomethyl);
C8(Carboxymethyl)
N-Term(TMT6plex); C7(Carbamidomethyl);
C8(Carboxymethyl)
N-Term(TMT6plex); C9(Carboxymethyl)
N-Term(TMT6plex); C6(Carboxymethyl); K21(TMT6plex)
N-Term(TMT6plex); C6(Carboxymethyl); K21(TMT6plex)
N-Term(TMT6plex); C6(Carboxymethyl); K21(TMT6plex)
Protein
Aging
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
RyR type 2, mus musculus
0,22313
0,47332
0,16294
-0,1032
Up
Down
0,1637
0,92236
0,32801
0,51632
-0,0071
-0,0418
0,45811
0,01926
0,80331
0,067
-0,0398
-0,0946
* p=0.002
(unpublished data)
Proteomic analysis identified increased glycation of RyR at
different peptides in the myocardium of aged mice
Advanced glycation end-products (AGEs) and RyR in mouse
cardiomyocytes
YOUNG
OLD
% of colocalization
between RyR and AGEs
100
80
RyR
RyR
60
*
40
20
0
AGE
Colocalizing spots
AGE
Colocalizing spots
10 µm
4-6 months ≥20 months
(unpublished data)
Aging is associated with increased colocalization between AGEs and RyR
Summary and conclusion
Experimental studies show SR-mitochondria disruption and
bioenergetic mismatch, both in aging and failing cardiomyocytes.
In aging, glycation of RyR could be the cause of SR-mitochondria
disruption and SR calcium leak.
Chronic exposure of mitochondria to leaky SR may underlie
increased mitochondrial calcium content, bioenergetic mismatch
and enhanced susceptibility to mPTP in aged cells.
SR-mitochondria disruption may facilitate the
transition from health to disease in the senescent
heart
Lab. of Cardiology, Vall d’Hebron
Institut de Recerca, Barcelona
Elisabet Miro-Casas, ScD
Marta Minguet, ScD
David Alujas, ScD
Laura Valls, ScD
Angeles Rojas, Technician
Ursula Vilardosa, Technician
Jose Castellano, PhD
Javier Inserte, PhD
Ignasi Barba, PhD
Antonio Rodriguez-Sinovas, DVM PhD
Marisol Ruiz-Meana, DVM PhD
Centro Nacional de Investigación
Cardiovascular (CNIC), Madrid
Celia Castañs, ScD
Elena Bonzón, PhD
Prof. Jesús Vázquez
Prof. David Garcia-Dorado
FUNDING
Suported by the Spanish Ministry of Science (SAF 2008-03067), Instituto de Salud Carlos III
(RETICS-RECAVA RD06/0014/0025 and FIS-PI15/1655) and Fundació MTV3.
Glucose
glucose-6-P
Glycolysis
fructose-6-P
fructose-6-diP
glyceraldehyde-3P
Glutathione
S-D-lactoyl
glutathione
GLO-2
D-lactate
Glutathione
GLO-1
methylglyoxal
AGEs
(protein glycation)
pyruvate
Anaerobic
glycolysis(hypoxia)
Krebs
L-lactate
Study design-II
Isolation of mitochondria
(protease digestion + differential centrifugation)
In vitro assay of mitochondrial respiratory
complex activity (spectrophotometry)
O2 consumption in intact
mitochondria (oxymetry)
290
State 2
nmols O/mL.min
270
ADP 250µM
250
230
substrates
State 2: O2 consumption with specific substrates
State 3: ADP-stimulated O2 consumption
Respiratory control ratio (RCR): State 3/State 2
State 3
210
State 4
190
170
Variable of interest: Statistical analysis:
HF (NYHA≥2) and AGE
multivariate ANOVA
Confounders: Demographics / Risk factors/
Comorbidities/ Treatment/ Type of surgery
150
0
100
200
300
400
Time (s)
figure 1. Methylglyoxal: Changing the face of the Proteome and the Genome
Many factors contribute to the accumulation of methylglyoxal, including hyperglycemia, uremia, oxidative stress, aging, and inflammation.
Methylglyoxal can react with and modify both proteins and DNA, leading to the generation of advanced glycation end products (AGEs). The
modification of targets by methylglyoxal and its derivatives contributes to upregulation of inflammatory and tissue-injury-provoking molecules (at least
in part through the interaction of AGEs with their receptor RAGE), gene transcription, protein crosslinking, and apoptosis. In this issue of Cell,
Brownlee and colleagues (Yao et al., 2006) report a new role for methylglyoxal in the modulation of gene expression through glycation of critical
arginine residues in the corepressor protein mSin3A. Understanding the biology of methylglyoxal may shed light on conditions and processes as
diverse as diabetes, uremia, cancer, and aging
A)
B)
CML
GLO-1
500
*
300
8
µmol/g
µg CML-BSA/g
400
10
6
200
4
100
2
0
0
<75 years ≥75 years
*
<75 years ≥75 years
D)
C)
Total GSH
400
600
300
400
200
*
µmol/g
µmol/g
800
200
100
0
0
<75 years ≥75 years
D-lactate
*
<75 years ≥75 years