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Presenter Disclosure
Shaina R. Eckhouse, M.D.
The following relationships exist related to this presentation:
Shaina Eckhouse: NIH NRSA
Christine B. Logdon: none
J. Marshal Oelsen: none
Elizabeth C. O’Quinn: none
Adam W. Akerman: none
Robert E. Stroud: none
Rupak Mukherjee: none
Jeffrey A. Jones: VA Merit Award
Francis G. Spinale: NIH grant, VA
Merit Award, MicroVide
DIFFERENTIAL MEMBRANE TYPEDIFFERENTIAL MEMBRANE TYPE--1 MATRIX 1 MATRIX
METALLOPROTEINASE SUBSTRATE PROCESSING METALLOPROTEINASE SUBSTRATE PROCESSING
WITH ISCHEMIAWITH ISCHEMIA--REPERFUSION: RELATIONSHIP REPERFUSION: RELATIONSHIP
TO INTERSTITIAL MICRORNA DYNAMICS AND TO INTERSTITIAL MICRORNA DYNAMICS AND
MYOCARDIAL FUNCTIONMYOCARDIAL FUNCTION
Shaina R. Eckhouse, Christine B. Logdon; J. Marshal
Oelsen; Elizabeth C. O’Quinn; Adam W. Akerman; Robert E.
Stroud; Rupak Mukherjee; Jeffrey A. Jones, Francis G. Stroud; Rupak Mukherjee; Jeffrey A. Jones, Francis G.
Spinale
Division of Cardiothoracic Surgery, Medical University of South Carolina,
Charleston, South Carolina, Ralph H. Johnson Veterans Affairs Medical
Center, Charleston, WJB Dorn Veterans Affairs Medical Center, Columbia,
and University of South Carolina School of Medicine, Columbia, South
Carolina
Myocardial Ischemia ReperfusionMyocardial Ischemia Reperfusion
• Ischemia Reperfusion (I/R) Injury = Myocardial
dysfunction despite restoration of adequate
blood flow
– Acute Changes = Extracellular Matrix (ECM)
Degradative Pathways• Matrix Metalloproteinases (MMPs)
– Chronic Changes = ECM Profibrotic Pathways• Can lead to changes in left ventricular (LV) geometry
and function (LV remodeling)
LTBP-1
TGF-β1β1β1β1TGF-β1β1β1β1
3344
NucleusNucleus
MT1-MMP
Synthesis
MT1MT1--MMPMMP
mRNAmRNA
MT1MT1--MMPMMP
TranscriptionTranscription
11
22
Unresolved Issue #1Unresolved Issue #1
MT1-MMP activity and MT1-MMP mediated
LTBP-1 substrate processing have never been
simultaneously measured in vivo.
Hypothesis #1Hypothesis #1
I/R injury induces a change in MT1-MMP
substrate processing and stimulates
downstream profibrotic pathways.
MicrodialysisMicrodialysis
Active MMP
Substrate
Cleaved
Substrate
Interstitial SpaceInterstitial Space
Microdialysis Microdialysis
ProbeProbeMyocardiumMyocardium
Experimental DesignExperimental Design
I/R Region
• Interstitial Fluid was collected
every 30 minutes
• LV harvested after I/R protocol
• n=12 I/R pigs
• n=5 Referent control pigs
I/R Region
Caval
Occluder
Caval
Occluder
Experimental TimelineExperimental Timeline
SteadySteady--statestate
30 60 90 30 60 90 minutesminutes
IschemiaIschemia ReperfusionReperfusion LV LV
harvestharvest
1200 120
MicrodialysisMicrodialysis ResultsResults
MT1MT1--MMPMMP
% change from Steady-state
100
150
200
% change from Steady-state
100
150
200
*
*
**
*
I/R Region Remote Region
SS 30 60 90 30 60 90 120
% change from Steady
0
50
SS 30 60 90 30 60 90 120
% change from Steady
0
50
Ischemia Reperfusion
Timepoints
Ischemia Reperfusion
Timepoints
*p<0.05 compared to SS
ANOVA p<0.05
ANOVA p<0.05
**
MicrodialysisMicrodialysis ResultsResults
LTBPLTBP--11
% change from Steady-state
100
150
% change from Steady-state
100
150I/R Region Remote Region
SS 30 60 90 30 60 90 120
% change from Steady
0
50
SS 30 60 90 30 60 90 120
% change from Steady
0
50ANOVA p<0.05
*
Ischemia Reperfusion
Timepoints
Ischemia Reperfusion
Timepoints
*p<0.05 compared to SS
Activation of TGFActivation of TGF--ββ SignalingSignaling
pSMAD2
Remote I/RControlControl Remote I/R
SMAD2
Percent Change of Control (%
)
500
600
Percent Change of Control (%
)
500
600* I
* p<0.05 compared to Control
p<0.05 compared to Remote RegionI
Control Remote I/R
Percent Change of Control (%
)
100
200
300
400
500
* I
Control Remote I/R
Percent Change of Control (%
)
100
200
300
400
500
*
Summary #1Summary #1
• Myocardial I/R resulted in acute MT1-MMP
activation
• Increased LTBP-1 substrate processing only • Increased LTBP-1 substrate processing only
within the I/R region acutely
• Early activation of TGF-β signaling pathway
with I/R
– Increased TGF-β profibrotic pathway over time
I/R Injury
Increased ECM
Proteolysis
Late PhaseEarly Phase
Chronic
LV
RemodelingIncreased
TGF-β
Profibrotic
Pathway
Upstream
Regulation
microRNA
(miRs)
Remodeling
microRNAmicroRNA and two possible courses and two possible courses
of actionof action
Inhibit mRNA Translation Cellular
Exportation
Relevant to Myocardial Fibrosis
miR-133a
miR-29a
Fibroblast Cell Culture
↑↑ miR-133a: ↓↓ MT1-MMP
↓↓ miR-133a: ↑↑ MT1-MMP
Putative miR-133a Targets
TGF-β Receptor I MT2-MMP
TGF-β2 MMP-9
Type I Collagen LTBP-1
CTGF
Unresolved Issue #2Unresolved Issue #2
However, direct in vivo interrogation of miRs in
the myocardial interstitium as a measure of
cellular levels remains unexplored.
Hypothesis Hypothesis #2#2
Dynamic and differential changes in cellular
export of miRs-133a and 29a occurs in
response to I/R.
Total Myocardial miR Levels Total Myocardial miR Levels (Intracellular + Interstitium)(Intracellular + Interstitium)
miR-133a
Change in m
iR Fold Expression
(ddCT)) 1.0
1.2
1.4
1.6
miR-29a
Change in m
iR Fold Expression
(ddCT)) 1.0
1.2
1.4
1.6
I/R Region
Remote Region
I/R Region Remote Region
Change in m
iR Fold Expression
(2^-(ddCT))
0.0
0.2
0.4
0.6
0.8
I/R Region Remote Region
Change in m
iR Fold Expression
(2^-(ddCT))
0.0
0.2
0.4
0.6
0.8
Interstitial Interstitial miRmiR Levels Levels (Extracellular)(Extracellular)
0.004
0.005
0.006
0.007
29a Expression Levels
Steady-state
miR-29a
133a Expression Levels
0.004
0.005
0.006
0.007
I
miR-133a
Steady-state
133a Expression Levels
0.004
0.005
0.006
0.007
I
miR-133aSteady
0.004
0.005
0.006
0.00729a Expression Levels
Steady
miR-29a
I/R Region
Remote Region*p<0.05 compared to Steady-state
p<0.05 compared to IschemiaI
Ischemia Reperfusion
0.001
0.002
0.003
Interstitial miR-29a Expression Levels
state ±
SEM
Interstitial miR-133a Expression Levels
Ischemia Reperfusion
0.001
0.002
0.003
**
*
state ±
SEM
Interstitial miR-133a Expression Levels
Ischemia Reperfusion
0.001
0.002
0.003
0.004
**
*
Steady-state ±
SEM
Ischemia Reperfusion
0.001
0.002
0.003
0.004
Interstitial miR-29a Expression Levels
Steady-state ±
SEM
ConclusionsConclusions
• Acute activation of PROTEOLYTIC and
PROFIBROTIC Pathways
• Differential and dynamic alterations in miR-133a • Differential and dynamic alterations in miR-133a
exportation into the myocardial interstitium
– Acute changes in miR-133a may regulate chronic
translation of MT1-MMP
– Identifies miR-133a as a potential target for regulating
myocardial fibrosis over time
Cardiothoracic Surgery Research at Cardiothoracic Surgery Research at
MUSCMUSC
CARDIOLOGY
Michael R. Zile, MD Catalin Baicu, PhD
Paul J. McDermott, PhD Amy Bradshaw, PhD
PEDS CARDIOLOGY
Eric M. Graham, MD J. Philip Saul, MD
Andrew M. Atz, MD
ANESTHESIOLOGY
Scott T. Reeves, MD Tamas A. Szabo, MD, PhD
James H. Abernathy, MD Ilka D. Theruvath MD, PhD
Francis McGowan, MD
CT SURGERY
John S. Ikonomidis, MD, PhD Jeffrey A. Jones, PhD
Francis G. Spinale, MD, PhD Rupak Mukherjee, PhD
Robert E. Stroud, MS William M. Yarbrough, MD
J. Matthew Toole, MD Stacia DeSantis, PhD
Paul J. McDermott, PhD Amy Bradshaw, PhD
Michael R. Gold, MD, PhD Donald R. Menick, PhD
Andrew M. Atz, MD
Cardiothoracic Surgery
Research Laboratory
Fellows/Medical Students
Jennifer Dixon, MD Shaina Eckhouse, MD
Foster Gaillard
Research Staff
Nikole O’Quinn Heather Smith
Risha Patel Chrissy Bond
Adam Akerman J. Marshal Oelsen
Allison Rice Billy Rawls
CT Research
Laboratory Dedicated to
Horace B. Smithy, MD
1914 - 1948
Regional Preload Recruitable Stroke Regional Preload Recruitable Stroke
Work (rPRSW)Work (rPRSW)
rPRSW (mmHg)
100
120
140
160
*
Timepoints (minutes)
BL 30 60 90 30 60 90 120
rPRSW (mmHg)
20
40
60
80
100
Ischemia Reperfusion
*
*
** * *
*
*
p<0.05 versus Baseline (BL)*
I/R Region
Remote Region
Simulated Ischemia/ReperfusionSimulated Ischemia/Reperfusion
miR-133a% Change from Norm
oxia
80
100
120
140
*
Normoxia Hypoxia Reoxygenation
% Change from Norm
oxia
20
40
60
80 *
*p<0.05 compared to Normoxia
Translational
Regulation
Exportation
The Synthesis and The Synthesis and
Interactions of Interactions of
microRNAsmicroRNAs
2 possible
interactions
Relevant to Myocardial Fibrosis
miR-133a
miR-29a