1. Journal of Cardiovascular
Pharmacology and Therapeutics
Intramyocardial Injection of Heart TissueDerived Extracellular Matrix Improves
Post-infarction Cardiac Function in Rats
Wangde Dai, MD, Paul Gerczuk, MD Yuanyuan Zhang, MD,
Leona Smith, MD, Oleg Kopyov, MD, Gregory L. Kay, MD,
Aarne J. Jyrala, MD, and Robert A. Kloner, MD, PhD

2. Introduction
Ischemic injury
myocyte necrosis
fibrotic scar tissue
breaking down extracellular matrix(ECM)
myocyte slipping
ventricular wall thinning &dilation
infarct expansion

3. Introduction
ventricular wall thinning &dilation
Laplace’s law
post-myocardial infarction
thickness
Tension

4. Introduction
Goal: thicker the ventricle
methods:
•Stem cell
•Acellur injection
decellularized ECM
1.Small intestine
2.Pericardium
3.Myocardial tissue

5. Introduction
Implantation of heart-tissue-derived ECM
Left Ventricular wall (LV wall)
•vascularization
•c-kit+ cell
•paradoxical LV sytolic bulging
•LV function

6. Material & Method
• Rat Heart-derived decellularized ECM
• Surgical Procedures for coronary ligation
&matrix injection
• Assessment of cardiac function by
Echocardiography and Left Ventriculography
• Measurement of hemodynamics
• Assessment of postmortem LV volumes &
histological parameters
• Immunohistochemical C-kit staining

7. Rat Heart-derived decellularized ECM
Fisher rat’s heart
T10 basic ULTRA-TURRAX Disperser
T10 basic ULTRA-TURRAX Disperser
Heart sctions
Heart sctions
3.4 mol/L NaCl2 solution
3.4 mol/L NaCl2 solution
““three 30-minute treatments””
three 30-minute treatments
cut into 1-mm
thick sections
50ml conical tube
50ml conical tube
Heart sections
Heart sections
PBS+1% antibiotic antimitiotic solution
PBS+1% antibiotic antimitiotic solution
““rinse”
rinse”
centrifuged at 3000 rpm
centrifuged at 3000 rpm
for 10 min
for 10 min
4ºC Shaker 1hour
4ºC Shaker 1hour
Heart tissue sample
Heart tissue sample
PBS nuclease solution
PBS nuclease solution
(50 units/mL DNAse + 10mg/mL RNAse)
(50 units/mL DNAse + 10mg/mL RNAse)
““Decellularization”
Decellularization”

8. Rat Heart-derived decellularized ECM
centrifuged at 3000 rpm
centrifuged at 3000 rpm
for 10 min
for 10 min
4ºC Shaker 1hour
4ºC Shaker 1hour
Heart tissue sample
Heart tissue sample
1% Triton X-100
1% Triton X-100
PBS solution
PBS solution
wash 3 times
wash 3 times
(1 hour per washing)
(1 hour per washing)
Heart ECM pellets
Heart ECM pellets
Stored at -80ºC
Stored at -80ºC

9. Surgical Procedures for coronary ligation &matrix injection
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)
intubated
intubated
Thoracotomy -- heart exposed
Thoracotomy heart exposed
Left coronary artery ligated
Left coronary artery ligated
Chest closed (suturing muscle & stapling skin)
Chest closed (suturing muscle & stapling skin)
Bupreax (0.001 mg/100 g) sc for 2days twice daily
Bupreax (0.001 mg/100 g) sc for 2days twice daily
Recover for aa week
Recover for week

10. Surgical Procedures for coronary ligation &matrix injection
Recover for aa week
Recover for week
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)
intubated
intubated
Thoracotomy -- heart exposed
Thoracotomy heart exposed
Matrix(75 µL) or saline (75 µL) directly injected in to the LV
Matrix(75 µL) or saline (75 µL) directly injected in to the LV
infarcted area
infarcted area
Recover for 6 weeks
Recover for 6 weeks
II am ready!
am ready!

11. Assessment of cardiac function by Echocardiography
Sono 5500 ultrasound system, 15MHZ transducer
Sono 5500 ultrasound system, 15MHZ transducer
Motion-mode(M-mode)
Motion-mode(M-mode)
FS (%) = [[ (EDD-ESD) // EDD ]*100%
FS (%) = (EDD-ESD) EDD ]*100%
FS: Frictional shortening
FS: Frictional shortening
3 consecutive beats and
3 consecutive beats and
averaged
averaged
global LV function
global LV function
EDD: The diameter across aa ventricle at the
EDD: The diameter across ventricle at the
end of diastole (End-diastolic dimension)
end of diastole (End-diastolic dimension)
ESD: similar to the end-diastolic dimension, but
ESD: similar to the end-diastolic dimension, but
is measured at the end of systole (End-systolic
is measured at the end of systole (End-systolic
dimension)
dimension)

12. Assessment of cardiac function by Left ventriculography
Xi Scan 1000 C-arm X-ray System
Xi Scan 1000 C-arm X-ray System
catheter inserted into left jugular vein
catheter inserted into left jugular vein
and injected 1 mL nonionic contrast
and injected 1 mL nonionic contrast
Both anterior-posterior and lateral video
Both anterior-posterior and lateral video
images acquired. 30frames/s
images acquired. 30frames/s
EF(%)=[ (ED_LV_Volumn -- ES_LV_Volumn) ]] //
EF(%)=[ (ED_LV_Volumn ES_LV_Volumn)
ED_LV_Volumn
ED_LV_Volumn
EF: LV ejection fraction
EF: LV ejection fraction
3 consecutive beats and
3 consecutive beats and
averaged
averaged
global LV function
global LV function

13. Assessment of cardiac function by Left ventriculography
Xi Scan 1000 C-arm X-ray System
Xi Scan 1000 C-arm X-ray System
catheter inserted into left jugular vein
catheter inserted into left jugular vein
and injected 1 mL nonionic contrast
and injected 1 mL nonionic contrast
Both anterior-posterior and lateral video
Both anterior-posterior and lateral video
images acquired. 30frames/s
images acquired. 30frames/s
Paradoxical systolic bulging(%) = (( circumference of the
Paradoxical systolic bulging(%) = circumference of the
bulging segment // total LV systolic circumference )) *100%
bulging segment total LV systolic circumference *100%
3 consecutive beats and
3 consecutive beats and
averaged
averaged
global LV function
global LV function
ES tracing extend out side the ED tracing
ES tracing extend out side the ED tracing

14. Measurement of hemodynamics
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)
2F high-fidelity, catheter-tipped micronanometer inserted into carotid artery
2F high-fidelity, catheter-tipped micronanometer inserted into carotid artery
record arterial blood pressure and heart rate
record arterial blood pressure and heart rate

15. Assessment of postmortem LV volumes
Unisperse blue dye 0.6mL 50% stain the Blood
Unisperse blue dye 0.6mL 50% stain the Blood
Vessel in the LV wall
Vessel in the LV wall
euthanized with 1mL KCL (149mg/mL)
euthanized with 1mL KCL (149mg/mL)
Harvest the heart
Harvest the heart
LV Volumes = Water in the cavity
LV Volumes = Water in the cavity

16. Assessment of Postmortem Histological Parameters
Hearts cut into 3 transverse slices
Hearts cut into 3 transverse slices
Middle slice for histology
Middle slice for histology
sectioned into 5µm thickness
sectioned into 5µm thickness
stained with H&E and Picrosirius red
stained with H&E and Picrosirius red
Unisperse blue dye 0.6mL 50% stain the
Unisperse blue dye 0.6mL 50% stain the
Blood Vessel in the LV wall
Blood Vessel in the LV wall
Blood vessel density
Blood vessel density
10 microscopic fields at x400 at infarct
10 microscopic fields at x400 at infarct
area choose randomly for counting the
area choose randomly for counting the
blue particles
blue particles
scar thickness
scar thickness
Computerized planimetry tracing
Computerized planimetry tracing
LV cavity area and total
LV cavity area and total
LV area
LV area
total LV epicardial circumference and
total LV epicardial circumference and
endocardial circumference
endocardial circumference
LV infarcted segment epicardial circumference
LV infarcted segment epicardial circumference
and endocardial circumference
and endocardial circumference

17. Immunohistochemical C-kit Stain
Slices stained with antibodies against c-kit (a marker for stem cell)
Slices stained with antibodies against c-kit (a marker for stem cell)
Slices
Slices
EDTA PH9.0
EDTA PH9.0
““epitope retrieval””
epitope retrieval
Cardioprotective c-kit+ cells are from the bone
Cardioprotective c-kit+ cells are from the bone
marrow and regulate the myocardial balance of
marrow and regulate the myocardial balance of
angiogenic cytokines.
angiogenic cytokines.
0.01% Triton
0.01% Triton
““permeabilized”
permeabilized”
rabbit anti-c-kit
rabbit anti-c-kit
primary antibodies
primary antibodies
4ºC over night
4ºC over night
biotinylated horse antibiotinylated horse antirabbit-secondary
rabbit-secondary
antibodies
antibodies
Image Pro Plus Software
Image Pro Plus Software
““Quantification”
Quantification”

18. Results
The LV FS by Echocardiography
Saline (n=17)
Matrix (n=19)
Before coronary
occlusion
47.3%±1.3%
46.5%±0.9%
After coronary
occlusion
1 week
21.4%±1.6%
20.7%±2%
After coronary
occlusion
6 week
17.8%±1.5%
26.2%±2.2%

19. Results
The LV FS by Echocardiography
Figure 2. Matrix injection significantly improved the left
ventricular(LV) fractional shortening compared to saline at 6 weeks
after treatment(P=0.0034).

20. Results
The LV EF and Paradoxical Systolic Bulging by LV Ventriculography
Figure 3. A: left ventricular ejection fraction (LVEF) calculated by angiography at 6 weeks after matrix or saline injection directly into
the scar area of myocardial infarction in rats. Matrix significantly increased the LVEF (P=0.043). Panel B: The extent of paradoxical
systolic bulging is expressed as (circumference of the bulging segment/total LV systolic circumference) *100%. Matrix implantation
significantly prevented paradoxical LV systolic bulging in the matrix-treated group compared to the saline-treated group (P =0.048).

21. Results
Hemodynamics
No significant differences were noted in heart
No significant differences were noted in heart
rate, systolic and diastolic blood pressure
rate, systolic and diastolic blood pressure

22. Results
Postmortem LV Volumes, Scar Thickness, Infarct Sizes, and Expansion Index
Scar thickness significantly increase
Scar thickness significantly increase
Infarct expansion significantly decrease
Infarct expansion significantly decrease

23. Results
Postmortem LV Volumes, Scar Thickness, Infarct Sizes, and Expansion Index
Figure 5. Scar thickness, average of the measurements at 5
equidistantpoints of the infarcted left ventricular wall, is significantly
higherin the matrix group compared to the saline group (P =0.0084).

24. Results
Blood Vessel Density and Recruitment of Endogenous Stem Cells in Infarcts
Saline
(n=17)
BV density in
scar area
(vessels/mm2)
Matrix
(n=19)
189±12
165±23
P=0.077 (>0.05 No significant difference)

25. Results
Blood Vessel Density and Recruitment of Endogenous Stem Cells in Infarcts
Saline
(n=6)
The number of
c-kit+ cells
Matrix
(n=6)
105±13
111±11
Similar!!
Figure 6. Immunohistochemical staining with primary
antibody against c-kit of the rat hearts (A) control
received saline and (B) received matrix implantation. The
red arrows identify positive c-kit stained cells (brown
color stained) within he infarct area(magnification 400,
scale bar . 20 mm).

26. Discussion
Biomaterial Implantation Has Similar Effects to That of Cell
Biomaterial Implantation Has Similar Effects to That of Cell
Therapy for Myocardial Infarction
Therapy for Myocardial Infarction
LV infarct wall thickened
LV infarct wall thickened
Paradoxical systolic bulging decreased
Paradoxical systolic bulging decreased
LV EF enhanced
LV EF enhanced
For the FIRST TIME!
For the FIRST TIME!
ECM can do this!
ECM can do this!

27. Discussion
New Blood Vessel Formation Within the Implanted Biomaterials
New Blood Vessel Formation Within the Implanted Biomaterials
In our present study, injected materials did not
In our present study, injected materials did not
promote neovascularization.
promote neovascularization.
Our study suggests that the cardiac ECM that we
Our study suggests that the cardiac ECM that we
utilized prevented LV remodeling without
utilized prevented LV remodeling without
requiring an increase in neovascularization
requiring an increase in neovascularization

28. Results
Blood Vessel Density and Recruitment of Endogenous Stem Cells in Infarcts
Saline
(n=17)
BV density in
scar area
(vessels/mm2)
Matrix
(n=19)
189±12
165±23
P=0.077 (>0.05 No significant difference)

29. Discussion
Recruitment of Endogenous Stem Cells by Implanted Biomaterials
Recruitment of Endogenous Stem Cells by Implanted Biomaterials
Many studies proved that!
Many studies proved that!
There was no evidence that ECM enhanced recruitment
There was no evidence that ECM enhanced recruitment
of these cells above and beyond infarction alone.
of these cells above and beyond infarction alone.
Therefore, any benefit of the ECM cannot be attributed to an
Therefore, any benefit of the ECM cannot be attributed to an
increase in c-kit cells in our study
increase in c-kit cells in our study

30. Results
Blood Vessel Density and Recruitment of Endogenous Stem Cells in Infarcts
Saline
(n=6)
The number of
c-kit+ cells
Matrix
(n=6)
105±13
111±11
Similar!!
Figure 6. Immunohistochemical staining with primary
antibody against c-kit of the rat hearts (A) control
received saline and (B) received matrix implantation. The
red arrows identify positive c-kit stained cells (brown
color stained) within he infarct area(magnification 400,
scale bar . 20 mm).

31. Discussion
Biomaterial Implantation Prevents Post-infarction Paradoxical LV
Biomaterial Implantation Prevents Post-infarction Paradoxical LV
Systolic Bulging
Systolic Bulging
YES,
YES,
ECM can do this!
ECM can do this!

32. Conclusion
Implantation of heart-tissue-derived ECM
Left Ventricular wall (LV wall)
•paradoxical LV sytolic bulging
•LV EF function
Angiogenesis
Angiogenesis
Recruitment of endogenous
Recruitment of endogenous
stem cells
stem cells

33. Thanx for your listening