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Cardiomyopathies in
mitochondrial disorders
Anu Suomalainen Wartiovaara MD PhD, Professor
FinMIT
Research Program of Molecular Neurology Biomedicum-Helsinki University of Helsinki
http://research.med.helsinki.fi/neuro/Wartiovaara/default.htm
Mitochondrial disorders Progressive encephalopathy Neurodegeneration Strokes, demyelination Epilepsy, ataxia, parkinsonism, migraines cognitive decline, hemiparesis, psychiatric symptoms Cardiomyopathy, conduction defects Kidney dysfunction Diabetes Intestinal dysfunction: malabsorption, diarrhea Infertility, premature menopause Sideroblastic anemia Immunological defects
Deafness, hearing deficit Retinitis pigmentosa Optic neuropathy Cataracts Defects of vision, blindness Acute liver damage Hepatopathy Muscle weakness Cramps Tiredness in exercise Sensory or motor neuropathies Tingling, pain and numbness of extremities
Cardiac energy metabolism
➲ Over 90% of ATP synthesis from mitochondrial respira=on ➲Flexible use of all nutrients – glucose, fat, ketones ➲Despite large fluctua=ons in work load, amazingly stable metabolism -‐ “stability paradox”
Data analysis. The position of each pixel relative to the cellmust be identical for all three separately obtained images. Toavoid error arising from slight movement of the cell duringimage acquisitions, we aligned the three cell images, ifnecessary, before calculation of SMb as follows. First, we chosefive to seven marker points in a small region of interest (ROI)that were peculiar with respect to light absorption, and theabsolute positions of these points were recorded. Thesemarker points appeared to correspond to locations ofmitochon-dria according to the results of rhodamine-123 staining (datanot shown). We then checked whether these marker pointswere in fact found at the same absolute coordinates in theremaining two images. If not, the deflections of the markersfrom the expected coordinates were calculated. Finally, theaffine transform (linear mapping of an image including shiftsand rotation) was conducted over the remaining two cellimages so that the square errors of the deflections could beminimized. Also, the images were low-pass filtered threetimes.
After the alignment and low-pass filtering, the calculationdepicted in Eq. 4 was conducted for all the pixels. Wedetermined the local SMb as follows. First, we arbitrarilyselected, in the SMb image, �12 ⇥ 4-µm rectangular ROIswithin a cell parallel to the long axis of the cell (see Fig. 3).AnSMb distribution histogram was then calculated for each ROI.The histogram was subsequently fitted to a normal distribu-tion using IgorPro data analysis software (WaveMetrics,Lake Oswego, OR). In cases in which the histogram wassignificantly skewed or the standard deviation of the SMbhistogram was ⌃0.5, we discarded the data. Finally, wecalculated the mean of the histogram and regarded it torepresent the local SMb.
Calibration. We conducted a calibration that relates localSMb to local PO2. We added 2 mM NaCN to the suspensionmedium and conducted SMb measurements for superfusiongas containing either 0.25, 0.51, 0.96, 2.09, or 3.14% oxygen.We assumed that NaCN abolishes the consumption of oxygenby the cell, thereby abolishing PO2 gradients from the extra-cellular medium to the intracellular space. Hence, intracellu-lar PO2 is in equilibrium with gas PO2. The relationshipbetween PO2 of the superfusion gas and measured SMb wasfitted to the Hill equation.
V̇O2 measurement. Because the magnitude of intracellularPO2 gradients would be proportional to flux of oxygen to thecell (10), we used 1 µM CCCP (an uncoupler of oxidative
phosphorylation) to amplify the intracellular PO2 gradients.Therefore, we needed to determine V̇O2 of the cell in thepresence of 1 µM CCCP. Five milliliters of the cell suspensionwere placed in the airtight measuring cuvette, in which anoxygen electrode (model 17026, Instrumentation Laboratory)was inserted. The cell suspension was vigorously stirredusing a magnetic stirrer. The time-dependent decrease in PO2of the suspension medium was recorded, and the rate of fall ofPO2 (�PO2/�t in Torr/min) was converted to the rate of oxygenconsumption (nmol O2 ·min⇤1 ·106 cells⇤1) using the followingequation
V̇O2 ⌅ ⇧w· (�PO2 /�t)/N ⇥ 106
where N is the number of cells per milliliter and ⇧w is thesolubility of oxygen in water (1.62 µmol · l⇤1 ·Torr⇤1 at 27°C).
RESULTS
We attempted a visualization of intracellular oxygenwith a subcellular spatial resolution in the presence ofan uncoupler of oxidative phosphorylation, 1 µM CCCP.When the cell suspension was superfused with 2.09(15.2 Torr) or 3.14% (22.8 Torr) O2 gas, intracellular SMbaveraged over the cell was �0.4–0.7 (corresponding to�1.9–8.7 Torr; see the results of calibration below)(Fig. 1, solid curve). These results indicate the presenceof large PO2 gradients in the extracellular medium,presumably resulting from the absence of the specificoxygen carrier myoglobin and the unstirred layer sur-rounding the cell surface (18). It should be noted thatintracellular oxygenation in this condition appearscomparable to the volume-averaged SMb reported in theworking cardiac cell in vivo (3, 5).
Figure 2 shows the representative data demonstrat-ing the visualization of intracellular oxygenation in asingle individual cardiomyocyte. For PO2 of the superfu-sion gas of 15.2 Torr, significant gradients of SMb fromthe sarcolemma toward the center of the cell weredemonstrated (indicated in pseudo colors). To quantita-tively analyze these radial heterogeneities of SMb, wecalculated SMb in small rectangular ROIs within a cell
Fig. 2. Representative data demon-strating reconstruction of SMb. Trans-mitted images of a single cardiomyo-cyte (left) were converted to an SMbimage (right) using Eq. 4. SMb is repre-sented in pseudo colors. The cell wasincubated with 1 µM CCCP to augmentintracellular PO2 gradients. PO2 of su-perfusion gas was 15.2 Torr. Cell bound-aries were manually traced.
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Takahashi et al., Am J Physiol Heart Circ Physiol. 1998;275:225-‐233.
Why are not all mitochondrial disorders involving the heart?
Typical manifesta=on:
hypertrophic cardiomyopathy Early onset
Specific adult disorders -‐ mtDNA
Molecular mechanisms of cardiomyopathies Novel tools for diagnosis
Case report: Infantile hypertrophic
cardiomyopathy Clinical First child of healthy parents Normal pregnancy, neonatal period 4 months:
hypertrophic cardiomyopathy, muscle weakness lactate levels 2-7mmol/l Brain MRI normal, EEG slightly slow
Rapidly progressive disease 10 months: death due to cardiac arrest
Respiratory chain Complex I, III and IV deficiency
in heart
Götz, Tyynismaa et al. Am J Hum Genet 2011
• Children and adults with verified or suspected mitochondrial disorder of unknown cause (n>50)
• Children with cardiomyopathies (n>150)
à 50-60 million short sequence fragments à 1.5% of the whole genome =exome (18,000 genes) Comparison to control genome: - Known variants vs mutations
Whole-exome sequencing
Sequence of all coding gene regions of the patient’s genome
Results of whole-exome sequencing in the patient
P2 All variants 65849 Unknown 6323 Map to gene 1549
Homozygous 43
Damaging 7 Mitochondrial 1
True disease causing mutation?
Presence in population Absent in over 3000 control subjects
Presence in other cardiomyopathy families Functional consequnces
Family 2: prenatal hypertrophic cardiomyopathy
o Prenatal: extrasystolia o At birth: poor condition,
cardiomyopathy, hyperlactacidemia, death at postnatal day 3
o Brother died in utero at week 40 o Compound heterozygosity for two
AARS2 mutations
AARS2 gene: infantile cardiomyopathy gene AARS2 – targets to mitochondria
Aminoacyl-tRNA synthetases - important for mitochondrial translation
Charges a tRNA with aminoacid Specific synthetases for each tRNA-aa pair; mitochondrial and cytoplasmic tRNA structure specific; recognition often based on anticodon; amino acid specificity lower
L155R affects architecture of the catalytic domain R592W predicted to affect tRNA binding at editing
domain
Liliya Euro
Acceptor stem of tRNA, minor groove contacts with positively charged recidues R592 at level of tRNA G2:U71
Amino acyl formation affected at catalytic site
Amino acylation defect
Reduced alanine incorporation to polypeptides
L155R
Editing defect
Misincorporation of amino acids (serine, glycine) into polypeptides
R592W
impaired mitochondrial protein synthesis
P1 P2
Clinical history (family II) • II/IV child, girl • normal pregnancy and birth • at 6 months of age aeer infec=on
(o==s) =red, cough • sudden death at emergency room
at age 7 months • autopsy: severe cardiac
enlargement, hypertrophic cardiomyopathy
• III/IV child, girl • normal pregnancy and birth • 4 months: liver fagy
degenera=on – e=ology unknown
• 9 months: cardiomyopathy
• now 13 years: – cardiomyopathy, no extreme
physical stress – selec=ve mu=sm – normal school, learning
difficul=es
Carroll et al. Hum Mutat 2013
CI + IV deficiency in heart
Sk.Muscle Heart
C P1 C P1
Sk.Muscle Heart
C P1 C P1
CI_39kDa
CII_70kDa
CIII_45kDa
CIV_COXI
CIV_COXII
CV_ATP_alpha
COMPLEX III
COMPLEX II
COMPLEX IV
COMPLEX I
C1 P1 C2
BN-PAGE SDS-PAGE
Exome sequencing results: mitochondrial ribosome mutated
Hetero-‐ zygote carriers
Homo-‐ zygote muta=on
wt
Ø A muta=on in MRPL44, in exon 2 Ø c. 467 T>G, L156R Ø nuclear encoded component of a large subunit of the mitochondrial ribosome
Figures by Pirjo Isohanni G/T G/G G/G T/T
G/T G/T
Conclusions: children
Mitochondrial translation defects may manifest primarily as early cardiomyopathy Our experience and literature: mostly fatal, but if the patient survives the critical phase, cardiomyopathy may stabilize after school age Exome analysis reveals new syndromes with variable clinical manifestations – not previously recognized as single-gene disorders Our experience: most children’s mitochondrial disorders are caused by rare variants in single or few families – even in genetic isolates
Gene search possible from a single patient Major progress in DNA diagnosis & research Confirms diagnosis & inheritance pattern Provides means for genetic counseling & research of pathogenic mechanism
MELAS Mitochondrial brain disease, muscle symptoms,
lactate acidosis, stroke-like episodes
In Finland carriers ~1:5000 – 1:10.000 Australia cohort ~ 1:200
Symptoms:
Maternal-inherited diabetes + hearing loss (MIDD)
Severe children’s encephalopathy Early brain infarcts Muscle weakness Cardiomyopathy
MELAS: tRNALeu(UUR) mutation m.3243A>G
Adult onset MELAS
The most common form of MELAS in Finland Diabetes (adult-onset, insulin-dependent) + Hearing loss + Hypertrophic cardiomyopathy
[page 32] [Cardiogenetics 2013; 3:e6]
Family 1 The proband (II:2) is a 39-year old male,
who presented at the age of 35 years with exer-cise-induced muscle pain, proximal leg weak-ness, dyspnea and sinus tachycardia 106/minand S3 in cardiac auscultation. He had beenprescribed diuretics due to swelling of theankles. Figure 2A shows the electrocardiogram(ECG). Serum pro-B-type natriuretic peptide(pro-BNP) concentration was elevated but tho-rax X-ray did not show pulmonary congestion.The main echocardiographic finding was non-obstructive left-ventricular hypertrophy (LVH;Figure 2A, Table 2). Doppler examination sug-gested elevated filling pressures. Magneticresonance spectroscopy of the brain showedlactate increase in the thalamus, but no neuro-logical signs or symptoms. Skeletal musclesample showed that 20% of muscle fibers werecytochrome-c-oxidase (COX)-negative, succi-nate dehydrogenase positive, strongly support-ing the diagnosis of mitochondrial dysfunctionalso underlying his cardiomyopathy. Familyhistory was negative for mitochondrial dis-eases or cardiomyopathy. During clinical fol-low-up, his cardiologic condition has remainedstable, without arrhythmias or dyspnea. Leftventricular walls have remained thickened, butthe early peak diastolic LV/mitral annularvelocity (E/Em) ratios have nearly normalized.
Family 2The proband (III:6) is a woman with dia-
betes mellitus, sensorineural hearing impair-ment and depression. At the age of 51 yearsshe developed acute pulmonary edema, hyper-tension and lactacidemia. Despite regularpulse and normal sized heart, serum troponinT and creatinine kinase-MB isoenzyme mass(CK-Mbm) levels were elevated (Table 1). ECGand echocardiography demonstrated LVH, withdiminished systolic function and restrictivehemodynamics (Figure 2B, Table 2). Coronaryangiography demonstrated 70% stenosis in theproximal left anterior descending coronaryartery (LAD), a total occlusion of middle LADwith collaterals from the right coronary artery,and 50% and 80% stenosis in first diagonalbranch of LAD (D1) and second diagonalbranch of LAD (D2), respectively. LV end dias-tolic pressure was 18 mmHg. Medical treat-ment of coronary artery disease was chosen.The diagnosis of mitochondrial contribution inhypertrophic CMP was strongly supported byhistological results of skeletal muscle biopsysamples, showing over 30% of COX-negativemuscle fibers, many of them also showing theragged-red fiber appearance, typical and diag-nostic for mitochondrial myopathy. In follow-up she developed episodes of cardiac decom-pensation induced by atrial flutter (ventricularresponse of 90/min) or fibrillation (~ 110/min)alternating with sinusbradycardia. Theseepisodes were accompanied with metabolic
Article
Figure 1. The family pedigrees. The index patients are marked with a black arrow. Blacksymbols indicate cardiac hypertrophy and signs or symptoms of heart failure; gray indi-cates subjects with increased relative wall thickness in echocardiography. MtDNA muta-tions were as followed: Family 1 – MELAS T3258C tRNA Leu(UUR); Family 2 and 3 –MELAS A3243G tRNA Leu (UUR); % indicates the people whose mutant mtDNA het-eroplasmy levels were studied, and the amount of mutant mtDNA in different tissues.
Figure 2. A) Patient II:2, family 1. Electrocardiogram (ECG) shows lateral t-inversions(left panel) and echocardiography (right panel) increased left ventricular wall thickness inparasternal long axis view (arrows); B) Patient III:6, family 2. ECG shows lateral q-wavesand abnormal r-peaks in the lateral chest leads (left panel), echocardiography (rightpanel) left ventricular hypertrophy (arrows). Apical view.
MELAS-‐muta=on and cardiomyopathy
Lee ventricular hypertrophy -‐ leading sign of disease
-‐ thickening of heart walls upon acute decompensa=on, especially septum
[page 32] [Cardiogenetics 2013; 3:e6]
Family 1 The proband (II:2) is a 39-year old male,
who presented at the age of 35 years with exer-cise-induced muscle pain, proximal leg weak-ness, dyspnea and sinus tachycardia 106/minand S3 in cardiac auscultation. He had beenprescribed diuretics due to swelling of theankles. Figure 2A shows the electrocardiogram(ECG). Serum pro-B-type natriuretic peptide(pro-BNP) concentration was elevated but tho-rax X-ray did not show pulmonary congestion.The main echocardiographic finding was non-obstructive left-ventricular hypertrophy (LVH;Figure 2A, Table 2). Doppler examination sug-gested elevated filling pressures. Magneticresonance spectroscopy of the brain showedlactate increase in the thalamus, but no neuro-logical signs or symptoms. Skeletal musclesample showed that 20% of muscle fibers werecytochrome-c-oxidase (COX)-negative, succi-nate dehydrogenase positive, strongly support-ing the diagnosis of mitochondrial dysfunctionalso underlying his cardiomyopathy. Familyhistory was negative for mitochondrial dis-eases or cardiomyopathy. During clinical fol-low-up, his cardiologic condition has remainedstable, without arrhythmias or dyspnea. Leftventricular walls have remained thickened, butthe early peak diastolic LV/mitral annularvelocity (E/Em) ratios have nearly normalized.
Family 2The proband (III:6) is a woman with dia-
betes mellitus, sensorineural hearing impair-ment and depression. At the age of 51 yearsshe developed acute pulmonary edema, hyper-tension and lactacidemia. Despite regularpulse and normal sized heart, serum troponinT and creatinine kinase-MB isoenzyme mass(CK-Mbm) levels were elevated (Table 1). ECGand echocardiography demonstrated LVH, withdiminished systolic function and restrictivehemodynamics (Figure 2B, Table 2). Coronaryangiography demonstrated 70% stenosis in theproximal left anterior descending coronaryartery (LAD), a total occlusion of middle LADwith collaterals from the right coronary artery,and 50% and 80% stenosis in first diagonalbranch of LAD (D1) and second diagonalbranch of LAD (D2), respectively. LV end dias-tolic pressure was 18 mmHg. Medical treat-ment of coronary artery disease was chosen.The diagnosis of mitochondrial contribution inhypertrophic CMP was strongly supported byhistological results of skeletal muscle biopsysamples, showing over 30% of COX-negativemuscle fibers, many of them also showing theragged-red fiber appearance, typical and diag-nostic for mitochondrial myopathy. In follow-up she developed episodes of cardiac decom-pensation induced by atrial flutter (ventricularresponse of 90/min) or fibrillation (~ 110/min)alternating with sinusbradycardia. Theseepisodes were accompanied with metabolic
Article
Figure 1. The family pedigrees. The index patients are marked with a black arrow. Blacksymbols indicate cardiac hypertrophy and signs or symptoms of heart failure; gray indi-cates subjects with increased relative wall thickness in echocardiography. MtDNA muta-tions were as followed: Family 1 – MELAS T3258C tRNA Leu(UUR); Family 2 and 3 –MELAS A3243G tRNA Leu (UUR); % indicates the people whose mutant mtDNA het-eroplasmy levels were studied, and the amount of mutant mtDNA in different tissues.
Figure 2. A) Patient II:2, family 1. Electrocardiogram (ECG) shows lateral t-inversions(left panel) and echocardiography (right panel) increased left ventricular wall thickness inparasternal long axis view (arrows); B) Patient III:6, family 2. ECG shows lateral q-wavesand abnormal r-peaks in the lateral chest leads (left panel), echocardiography (rightpanel) left ventricular hypertrophy (arrows). Apical view.
Conclusions: MELAS-‐ m.3243A>G
cardiomyopathy a common manifesta=on
May be provoked by physical stress May be asymptoma=c May manifest as acute arrhythmia with rapid progressive disease course May stabilize
à All MELAS-‐carriers: cardiology consulta=on recommended
Thanks to all the pa=ents and their families who contributed to our studies and helped to increase understanding of mechanisms of mitochondrial disease
From Euromit to Horizon 2020: how to reinforce mito interna=onal networks
We start to be good in diagnosis
Progress in understanding mechanism
à Emphasis on therapy
Mitochondrial disorders: variability is a challenge for therapy trials
Progressive encephalopathy Neurodegeneration Strokes, demyelination Epilepsy, ataxia, parkinsonism, migraines cognitive decline, hemiparesis, psychiatric symptoms Cardiomyopathy, conduction defects Kidney dysfunction Diabetes Intestinal dysfunction: malabsorption, diarrhea Infertility, premature menopause Sideroblastic anemia Immunological defects
Deafness, hearing deficit Retinitis pigmentosa Optic neuropathy Cataracts Defects of vision, blindness Acute liver damage Hepatopathy Muscle weakness Cramps Tiredness in exercise Sensory or motor neuropathies Tingling, pain and numbness of extremities
Emphasis on therapy: Op=mal treatment trial with pa=ents who are/have
• Age & gender matched
• Similar manifesta=ons • Similar gene=c background • Similar amount of mutant mtDNA • Similar stage of severity
• Placebo-‐controlled double-‐blind studies à Small pa=ent groups, significance compromised à Interna=onal collabora=on
Knowledge -‐ Coordina=on -‐ Applica=on
Na=onal registries à Informa=on of natural history of disease à Standardized data collec=on à Enrollment to therapy trials with large enough study groups
Priori=za=on of therapy trials based on preclinical evidence à “interna=onal coordina=on group” for mitochondrial disease and treatment – Europe, USA, Japan, Australia; Bethesda 2012
Horizon 2020
• Horizon 2020: The biggest EU research and innova=on programme ever: €80 billion of funding available over 7 years (2014 to 2020)
• “The goal is to ensure Europe produces world-‐class science, removes barriers to innova=on and makes it easier for the public and private sectors to work together in delivering innova=on.”
Horizon 2020 aims / health • Research of mechanisms:
improve understanding of the causes and mechanisms underlying health, healthy ageing and disease
• Diagnos=cs and treatment: improve our ability to monitor health and to prevent, detect, treat and manage disease
• models and tools for health and care delivery.
• support older persons to remain ac=ve and healthy
Rare disorders are a major health problem
Affec=ng fewer than 1:2000 of popula=on In EU: 6000-‐8000 dis=nct rare diseases affect 6-‐8% of the popula=on – between 27 and 36 million people à Most are lacking therapy
Special issues for rare diseases
• small and dispersed pa=ent popula=ons • nature of the therapies oeen highly specialized and novel
• Academia and company collabora=on thin • limited market for such therapies à low commercial return à Low interest of pharma companies for development
Rare diseases Interna=onal Rare Diseases Research
Consor=um (IRDiRC)
• Launched 2011 to strengthen interna=onal collabora=on
• Aims to deliver 200 new therapies for rare diseases and means to diagnose most of them by the year 2020
• Co-‐funded by member states • aims at understanding of
disease mechanisms and natural history of rare diseases
• objec=ve to develop new diagnos=c tools and treatments (preclinical, animal models, cell-‐ gene therapy)
• Transna=onal • 5-‐6.000.000 € per grant • Total budget 60.000.000
Rare diseases Interna=onal Rare Diseases Research
Consor=um (IRDiRC) • Co-‐funded by member states: state commitment • aims at understanding of disease mechanisms and
natural history of rare diseases • objec=ve to develop new diagnos=c tools and
treatments (preclinical, animal models, cell-‐ gene therapy)
• Transna=onal • 5-‐6.000.000 € per grant • Total budget 60.000.000
Mitochondrial disease consor=a poten=al for:
Animal models Coordina=on of trials for promising therapeu=c strategies
– MtDNA maintenance diseases – Respiratory chain diseases of childhood – Transla=on disorders
Deadline for applica=ons 2014-‐10-‐14 17:00:00 (Brussels local =me)
Registries Pa=ent trials àCollabora=on with industry
Pa=ent organiza=ons – strong and important impact
• Different countries – very different levels of organiza=on in different countries
• Raising awareness -‐ lobbying • Facilita=ng funding • Spreading informa=on – collec=ng pa=ents together with a common voice
• Contacts to both research and pharma • Peer support
EUROMIT 2014 – Tampere, Finland
Mitochondrial medicine from 1990
First parallel conference of scien=sts with pa=ents and their families and carers Aim to -‐ Generate pa=ent support – issues vary in different countries
-‐ Raise awareness -‐ Enhance contact between pa=ent organiza=ons in Europe
-‐ Train medical personnel