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

TGFTGF--ββ and and ProfibroticProfibrotic SignalingSignaling

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.

MethodsMethods

I/R Region

Caval

Occluder

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

Summary #3Summary #3

miR-133a

MT1-MMP

Ribosomes

mRNA

Steady-

State

Ischemi

a

MT1-MMP Activity

mRNA

Reperfusio

n