Characterizing mitochondrial dysfunction: ischemia and reperfusion during Total Knee

Arthroplasty

Characterizing mitochondrial dysfunction: ischemia and reperfusion during Total Knee

Arthroplasty

Ryan BoileauMentor: Austin Hocker

Hans Dreyer, PI

Dreyer Muscle

Physiology Lab

Introduction• What is TKA?

-The most common treatment for chronic osteoarthritis.

-Replaces the entire joint, a tourniquet is often

implemented during surgery (>95%)

• Relevant Statistics-Prevalence of surgery predicted to increase 7 fold by 2030 to 3.48 million primary TKAs annually-Osteoarthritis affects more than 60% of Americans 65 years of age or older

• Persistent muscle atrophy is the greatest clinical barrier following TKA

Question?What is the extent of mitochondrial dysfunction

during and immediately after TKA?

Hypothesis?Ischemia/reperfusion during TKA stimulates

expression of pro-apoptotic factors in muscle cells.

Methods

• 11 subjects (ages 68±3.73) • 7 females and 4 males• Gel electrophoresis followed by Western blotting• qPCR

43 ± 4 min. 16 ± 3 min.

Mitochondrial apoptotic pathwayIschemia

Bid

Bax xL

Bad

CytC

Caspase 9

APAF-1

Caspase 3

Apoptosis

CHOP

IRE1a

BIM

ER stress

Mitochondria

Bcl2Beclin-1

Main Steps:1) Ischemia causes

activation of Bid2) Bax dimerization causes

MOMP to open and CytC leakage

3) APAF-1 Caspase activation

4) Apoptosome formation5) Organized cell demise

Results for ischemiaIschemia

Bid

Bax xL

Bad

CytC

Caspase 9

APAF-1

Caspase 3

Apoptosis

CHOP

IRE1a

BIM

ER stress

Mitochondria

Bcl2Beclin-1

Beclin-1

Bid

Bim

Casp3

Casp9

IRE1a

BAD

BAK

BAX

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Change in Transcript Level Relative to Baseline

Fold change in transcript levels

Gene

of I

nter

est

Results for ischemiaIschemia

Bid

Bax xL

Bad

CytC

Caspase 9

APAF-1

Caspase 3

Apoptosis

CHOP

IRE1a

BIM

ER stress

Mitochondria

Bcl2Beclin-1

Beclin-1

Bid

Bim

Casp3

Casp9

IRE1a

BAD

BAK

BAX

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Change in Transcript Level (relative to Baseline)

Fold change in transcript levels

Gene

of I

nter

est

IRE1a

Casp3

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Change in Cytoplasmic Level (relative to Base-

line)

Fold change in protein levels

Gene

of I

nter

est

Results for reperfusionIschemia

Bid

Bax xL

Bad

CytC

Caspase 9

APAF-1

Caspase 3

Apoptosis

CHOP

IRE1a

BIM

ER stress

Mitochondria

Bcl2Beclin-1

Beclin-1

Bid

Bim

Casp3

Casp9

IRE1a

BAD

BAK

BAX

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Change in Transcript Level Relative to Baseline

Fold change in transcript levels

Gene

of I

nter

est

Results for reperfusionIschemia

Bid

Bax xL

Bad

CytC

Caspase 9

APAF-1

Caspase 3

Apoptosis

CHOP

IRE1a

BIM

ER stress

Mitochondria

Bcl2Beclin-1

Beclin-1

Bid

Bim

Casp3

Casp9

IRE1a

BAD

BAK

BAX

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Change in Transcript Level Relative to Baseline

Fold change in transcript levels

Gene

of I

nter

est

IRE1a

Bcl2

Casp3

Casp9

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Change in Protein Level Relative to Baseline

Fold change in protein levels

Gene

of I

nter

est

Summary of resultsIschemia

Bid

Bax xL

Bad

CytC

Caspase 9

APAF-1

Caspase 3

Apoptosis

CHOP

IRE1a

BIM

ER stress

Mitochondria

Bcl2Beclin-1

Beclin-1

Bid

Bim

Casp3

Casp9

IRE1a

BAD

BAK

BAX

Bim

Casp3

BAD

BAX

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Change in Transcript Level Relative to Baseline

Ischemia Reperfusion

IRE1a

Bcl2

Casp3

Casp9

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Change in Cytoplasmic Protein Level Relative to Baseline

Fold change in protein levels

Gene

of I

nter

est

# P-value <0.20* P-value <0.05

#

#

#

*

Preliminary results• Our data suggests that induction of the apoptotic pathway is

occurring during TKA

• In addition, our results also suggest that anti-apoptotic factors are elevated under ischemic conditions

• Finally, expression of pro-apoptotic factors are diminished during reperfusion

Future research• Quantifying the cellular stress allows us to determine the

efficiency of future therapies in decreasing muscle atrophy following TKA

-Determine contribution of the endoplasmic reticular stress-Determine contribution of the TKA (tourniquet) on mitochondrial function using permeabalized fibers and respirometry-Determine contribution of lysosomal stress• Supplement subjects with essential amino acids to attenuate

atrophy through the changes in anabolic and catabolic pathways • Determine miRNA changes associated with TKA surgery and

tourniquet use

AcknowledgementsThanks to Peter O’Day and the SPUR staff/program for supporting my research NICHD Summer Research Program NIH-

1R25HD070817 Thanks to my PI, Hans Dreyer, and mentor, Austin Hocker for letting me do scienceFinally, thanks to Irene Sogge, Chris Banek, and Karen Needham for their generosity throughout the Summer