mission to mars - duke universityprecisionmedicine.duke.edu/files/gpmf_bowles_02.23.2017.pdf ·...
TRANSCRIPT
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Mission to Mars:
The Heart of the Matter and (micro) Gravity of the Situation
Dawn Bowles PhDCo-Director, Duke Human Heart RepositoryAssistant Professor, Department of Surgery
[email protected] 23, 2017
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• Background-NASA HRP, evidence, risk, and gaps during prolonged manned space exploration
• Microgravity’s influence on the cardiac proteome
• Space Radiation- Ongoing studies on cardiovascular function and proteomics
• Combined Effects of Microgravity and Space Radiation- Future studies
Outline
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Breaking News
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Immediate Plans for Human Exploration beyond LEO
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NASA- Human Research Program (HRP)
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Human Research Program “ Elements”
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NASA’s Human Research Program (HRP)
• NASA is distinct from NIH as they are evidence based, (evidence books) evaluate risks and gaps in knowledge.
33 risks225 gaps1173 tasks
• Y/N answer- Is there a problem? Mechanism not needed.
• How do we fix the problem? Mechanisms need for countermeasure development
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Astronaut Risk of
Cardiovascular Disease
Alterations from Space
Flight
Microgravity
Life Style and Genetic Factors
Radiation
Physical and Emotional Stress (i.e. isolation,
claustrophobia)
Circadian Rhythm
Disturbances
Biological Stressors of Manned Space Exploration Contributing to CVD Development
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Microgravity and Microgravity Simulators
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Physiological Consequences of Microgravity
System Effect Risk
Bone Loss of bone mineral density (1-2% per month)
Increased risk of bone fracture.Calcification of soft tissues and kidney stone formation
Muscle Calf muscle volume ↓ 13%Peak power ↓32%
Insufficient muscle strength to perform emergency egress and post-landing EVA
Cardiovascular Venous shift to upper bodyPlasma Volume ↓ 17%Total blood volume ↓ 10%Decreased vasoconstrictor responses
Orthostatic intolerance when upright upon return to gravity, increasing fall riskHeart a muscle- insufficient muscle strength?
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ug 1xg
0 h
12 h
48 h
120 h
+ SILAC+ beads
ACell pellet
Cell lysis, protein digestion
LC/MS/MS
AUCQuantification(Protein total)
BSILAC Ratio
Quantification(Turnover)
The Influence of Microgravity on the Cardiomyocyte Proteome- Feger et al
Hypothesis: Cardiac dysfunction in astronauts in microgravity could be tied to protein differential expression or changes in protein turnover.
C D
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44%
25%
31%
No Change Over TimeSimilar Change Over TimeDissimilar Change Over Time
B
B
A
The Influence of Microgravity on the Cardiomyocyte Proteome –Feger et al
848 proteins were compared for abundance
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Time (hours)
0 12 48 120
1xg 1xg ug 1xg ug 1xg ug0
0.5
1
Pept
ide
RIA
The Influence of Microgravity on the Cardiomyocyte Proteome-Feger et al
359 peptides/ 196 proteins were compared for turnover
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The Influence of Microgravity on the Cardiomyocyte Proteome –Feger et al
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Table 3
Primary Protein Name Protein Description %CV FC @ 120 h (ug v 1xg)function
392351353 Myosin-13 9.9 3.5 Molecular motor
11693154 Platelet-activating factor acetylhydrolase IB subunit beta 11.5 2.7 Activity of RhoGTPases, actin polymerization
198442897 AFG3-like protein 2 10.3 2.5 Mitochondrial protein homeostasis9507135 Spectrin beta chain, brain 2 5.2 2.1 Actin binding293359790 Protein FAM179A-like isoform 1 3.6 1.9 Unknown function
62945328 Protein NipSnap homolog 2 11.9 1.8 Mitochondria, neg regulation of ATP citrate synthase activity
25742739 Long-chain-fatty-acid--CoA ligase 1 2.5 1.7 Activates breakdown of complex FA
48675862 Acyl-coenzyme A thioesterase 2, mitochondrial 2.0 1.7 Catalyzes hydrolysis of acyl-CoA to free FA and co enzyme A
392347468 Poly(rC)-binding protein 1 6.2 1.6 mrna import to mitochondria
56605722 Serine hydroxymethyltransferase, mitochondrial 6.2 1.6 Conversion l-serine to glycine, provides 1 carbon units to cell
113205496 Pyruvate dehydrogenase complex, component X 2.1 1.6 Mitochondrial, tether E3 dimers to E2 core
392341350 Glyceraldehyde-3-phosphate dehydrogenase-like 1.3 1.5 Breaks down glucose for energy and carbon
13994225 3-hydroxyacyl-CoA dehydrogenase type-2 8.6 1.5 Mitochondrial tRNA maturation
11968102 Ornithine aminotransferase, mitochondrial precursor 5.2 1.5 Processes excess nitrogen during protein breakdown
392351018 Sarcalumenin 3.2 1.5 Calcium buffering in SR410110929 Stress-70 protein, mitochondrial 3.1 1.5 Mitochondrial protein homeostasis209954804 Plastin-3 9.5 -24.3 EF-hand protein, bone?
293346882 Mitochondrial aspartate aminotransferase-like 18.0 -22.2metabolite exchange between mitochondria and cytosol, uptake of long chain free fatty acids
8394079 Proteasome subunit beta type-2 11.8 -15.4 ATP dependent proteolytic activity
157818179 Elongation factor 1-beta 5.3 -13.4 Translation elongation, gdp to gtp exchange
157787127 40S ribosomal protein S28 8.2 -12.6 Ribosomal protein, translation392348740 Laminin subunit beta-1 4.2 -12.2 ECM structural component
77993298 Translocon-associated protein subunit alpha precursor 5.3 -12.0 ER protein, ER UPR
12018252 Transketolase 14.2 -8.9 Pentose phosphate pathway
56744249 Reticulocalbin 3, EF-hand calcium binding domain precursor 4.9 -7.6 ER Calcium binding
40254781 Rab GDP dissociation inhibitor beta 15.7 -6.8 Regulates gdp/gtp exchange of most rab proteins, poly A RNA binding
392342369 60S ribosomal protein L30-like 3.9 -6.7 Ribosomal protein, translation6981326 Protein S100-A4 7.0 -6.5 Ca binding, polyA RNA binding61556832 Adenine phosphoribosyltransferase 13.5 -6.3 AMP biosynthesis salvage pathway
6981574 SPARC precursor 15.7 -6.2 Regulates cell growth, thru ECM and binds calcium
27665858 40S ribosomal protein S9-like 12.2 -5.9 Ribosomal structural protein
392346755 Ribosome-binding protein 1 14.7 -5.6 Mediates interaction between ribosome and ER
392347136 Collagen alpha-2(I) chain-like isoform 1 7.2 -5.5 Extracellular matrix13592133 Actin, cytoplasmic 1 3.7 -5.3 motility203097140 Myosin regulatory light chain RLC-A 9.0 -5.2 Muscle contraction392348865 Histone H2A.V-like 4.5 -4.7 Chromosomal binding77404180 Ras-related protein Rab-4A 12.5 -4.6 ATPase activator activity157819753 Reticulocalbin-1 precursor 13.3 -4.5 ER calcium binding61556967 Elongation factor 1-delta 9.3 -4.1 Transfer of aminoacyl-tRNA to ribosome6981672 Tropomyosin alpha-4 chain 13.0 -3.7 Muscle contraction6978589 Non-muscle caldesmon 5.7 -3.6 Muscle contraction157822227 60S ribosomal protein L12 7.8 -3.4 translation148747365 Heat shock protein HSP 90-beta 2.3 -3.4 Classic heat shock protein392356007 Actin, cytoplasmic 1-like 5.0 -3.3 Cell motility
9506845 Rab GTPase-binding effector protein 1 15.1 -3.2 Membrane trafficking, protein localization
157786744 Dihydropyrimidinase-related protein 2 4.5 -3.0 Cytoskeletal organization6981240 Myosin light chain 3 4.3 -2.5 Muscle contraction
124107592 Unconventional myosin-Ic 6.1 -2.3 Associated with transcriptionally active ribosomal genes
12083607 40S ribosomal protein S14-like 2.5 -2.2 translation56090293
Pyruvate dehydrogenase E1 component subunit beta, mitochondrial precursor 4.1 -2.1metabolism
219275589 Asparaginyl-tRNA synthetase, cytoplasmic isoform 2 3.5 -2.1 translation
293350511 M2 pyruvate kinase-like isoform 1 2.1 -2.0 metabolism155369650 Myosin light polypeptide 6 6.2 -1.8 Muscle contraction11968086 Ribosomal protein L4 5.1 -1.7 translation66730475 Tropomyosin beta chain 7.2 -1.6 Muscle contraction6981236 Myosin-9 4.8 -1.6 Cytoskeletal organization6981666 Troponin T, cardiac muscle 3.5 -1.5 Cardiac structure
The Influence of Microgravity on the Cardiomyocyte Proteome –Feger et al
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The Influence of Microgravity on the Cardiomyocyte Proteome –Feger et al
SummarySimulated microgravity alters expression of proteins involved in multiple biological functions in thecardiomyocyte, including, energetics, protein translation, metabolism, muscle contraction and structure,ER stress, and protein transport and folding.
Protein turnover is severely diminished in response to simulated microgravity.
Cell damage and death do not seem to play a significant role in the diminished protein turnover as thelack of change in LDH release, caspase-3 activity, and global ubiquitination suggest.
These data, in addition to the reduced amount of contractile proteins, suggest decreased translationcould be a mediator of microgravity-induced cardiac atrophy.
Future studies involve evaluation of over-expression and knock down of key candidate proteins usingviral vector technology, SILAC labeling, and evaluating influence on cardiac cell size and proteinturnover rates.
Use of other models of microgravity for countermeasure development.
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radiation induced heart disease
Atherosclerosis
Conduction disturbances
Valvular disease
Endocardial disease
Myocardial disease
Pericardial disease
Space Radiation Induced Cardiovascular Dysfunction – High Dose
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Space Radiation Induced Cardiovascular Dysfunction- Chronic Low Dose
Terrestrial Radiation Space radiation
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Data collected during the Mars Curiosity rover's cruise to the red planet show the radiation environment in deep space, away from the protection offered by Earth's atmosphere and magnetic field, is more than three times higher than a crew aboard the International Space Station experiences during the same period. (Credit: NASA)
Space Radiation Induced Cardiovascular Dysfunction- Chronic Low Dose
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Space Radiation Induced Cardiovascular Dysfunction- Chronic Low Dose
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ObjectiveTo acquire a mechanistic understanding of how thespace radiation environment alters cellular andmolecular processes that contribute to thedevelopment of cardiac dysfunction at the organ andorganism level.
Hypothesisthe proteomic signatures in the heart arising fromdifferent space radiation exposure scenarios will bedifferent and will correlate with the resulting type,onset, and magnitude of cardiovascular dysfunction.
Space Radiation Induced Cardiovascular Dysfunction- Chronic Low Dose
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1. To evaluate the physiological and functional changesoccurring over time to the murine cardiovascular systemfollowing exposure to different space radiation scenarios.
2. To correlate cardiovascular functional changes with molecular pathwayperturbations revealed by systematic and integrative analysis of multipleproteomics data sets (quantitative, dynamic, and post-translationalmodification proteomics) obtained from biological samples from theseexperiments.
Specific Aims
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Approaches
Incorporation by Time & 2 more
Group
0 1
Inco
rpor
atio
n
0
0.2
0.40.6
0.8
1
0
0.2
0.40.6
0.8
1
0
0.2
0.40.6
0.8
1
0
0.2
0.40.6
0.8
1
012
48120
Time
Legend
0
12
48
120
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Pre-radiation Echos
Follow up echos over time post exposure Invasive terminal Testing and tissue collection at 9 months post radiation
Radiation exposure @ 6 months
C57BL6
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Type and doses of radiation
gamma (50cGy, 100cGy, 200cGy),
oxygen (15cGy, 25cGy, 50cGy), Fe (15cGy, 25cGy, 50cGy).
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Common List of echo dataMeasurement Mode Parameter Units
A'PW Tissue Doppler Mode Velocity mm/s
E'PW Tissue Doppler Mode Velocity mm/s
IVRT PW Doppler Mode Time msMV A PW Doppler Mode Velocity mm/sMV Decel PW Doppler Mode Acceleration mm/s2MV Decel PW Doppler Mode Time msMV E PW Doppler Mode Velocity mm/s
Calculation Units
A'/E' none
E'/A' none
MV Area (simplified) mm²
MV E/A none
MV E/E' none
Measurement Mode Parameter UnitsIVS;d M-Mode Depth mmIVS;s M-Mode Depth mmLVID;d M-Mode Depth mmLVID;s M-Mode Depth mmLVPW;d M-Mode Depth mmLVPW;s M-Mode Depth mm
Calculation UnitsEF %FS %LV Mass mgLV Mass (Corrected) mgLV Vol;d uLLV Vol;s uL
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Future plans
• Continue mouse echos- expect functional changes at 9 months, 1st study informs timing of proteomics analysis
• New group of mice 250 radiation time in April 11th
and 12th
• Undertake metabolic labeling of mice-dynamic proteomics
• Fractionated doses• Mixtures of radiation to better mimic deep space
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Combined effects-future studies• RNNC +/- radiation (gamma, iron, oxygen), +/- microgravity, dynamic SILAC
Mouse +/- radiation, +/- hindlimb unloading, dynamic proteomics- AHA labeling
• LEO/ISS studies
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AcknowledgementsMicrogravity Space Radiation Combined Effects
Bryan J. FegerJ. Will ThompsonLaura G. DuboisReddy P. KommaddiMatthew W. FosterRajashree MishraSudha K. ShenoyYoichiro Shibata4,Yared H. KidaneM. Arthur MoseleyLisa S. Carnell
Muath BishawiMark DewhirstDillon IsaacMike WatsonRaj MishraMaragatha KuchibhatlaTony SlabaDennis AbrahamLan MaoKaren TerryPeter GuidaAdam RusekDLAR StaffBNL Staff
Steve BlattnigYaron BaracNenad BursacKen YoungLetitia HubbardCraig Henriquez
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Tissue Sharing Opportunites
1. Each hour of irradiation at BNL costs NASA > $10,0002. Rare and valuable mice with annotated cardiac function,
proteomic analysis3. DHHR Biobanking all organs4. Searching for collaborators, behavioral studies, vision studies5. 2017 ISBER talk