Characterizing mitochondrial dysfunction: ischemia and reperfusion during Total Knee Arthroplasty Ryan Boileau Mentor: Austin Hocker Hans Dreyer, PI Dreyer.
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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
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