1) The document describes several animal models used to study heart failure, including models that induce heart failure through myocardial infarction or pressure overload. 2) The left anterior descending coronary artery ligation model is commonly used in rats and mice to induce myocardial infarction, producing reductions in ejection fraction that mimic human heart failure. 3) Ischemia-reperfusion injury models, where the coronary artery is temporarily occluded then reperfused, are also used and can assess treatments administered prior to or after the ischemic event.

2. Adapted from Riehle et al, Cardiovascular Research 2019
COPD: chronic obstructive pulmonary disease LVEF: left ventricular ejection fraction
HFrEF: heart failure with reduced ejection fraction HFpEF: heart failure with preserved ejection fraction
Risk factors for the development of heart failure with reduced ejection fraction and for
the development of heart failure with preserved ejection fraction
Current treatments only slow the progression of the syndrome
Renal
Dysfunction
Aging
COPD
↓LVEF
Volume
Overload /
Mitral Valve
Regurgitation
Myocardial
Infarction /
Ischemic
Injury
Drug Toxicity
Risk Factors
(Obesity,
Diabetes,
Hypertension)
Inflammation
EF<40% (reduced)
HFrEF
EF=40-49%
(mid-range) HFmrEF
EF ≥50% Preserved
(HFpEF)
Diastolic
Dysfunction
↑Ventricular
Load

3. Riehle et al, Cardiovascular Research 2019
Rodent models of heart failure with reduced ejection fraction (HFrEF)
TAC (Mouse or Rat)
Reliable model to induce cardiac
hypertrophy and HF
I/R (Mouse or Rat)
Close to clinical scenario
MI (Mouse or Rat)
Reliable model to induce tissue
damage and HF
Potent stimulus to induce cardiac
hypertrophy
Drug is easy to administer
Isoproterenol (Mouse)
I/R- Ischemia Reperfusion
Animal Models

4. Acute Model
2 to 72 hours
Advantage
- Cost
- Fast
-Only acute measures
Disadvantage
- Always anesthetized
- Terminal
Chronic Model
2 to 16 weeks
Advantage
- Conscious
- Continuous measures
Disadvantage
- Higher Cost
Temporary Ligation Permanent Ligation
ANIMAL MODEL OF HF: ISCHEMIC INJURY

5. Rat and Mouse Model: Left Anterior Descending (LAD) Coronary Artery Ligation
Occlusion site
Temporary Ligation: Temporary ligature that can be
removed for timed reperfusion. The suture is placed under
the LAD coronary artery and tied to a PE tubing.
Reperfusion occurs by removing the tubing.
Permanent Ligation: The ligature placed under the LAD
coronary artery and tied.
LAD- Left anterior descending

6. Adapted from French et al Adv Drug Deliv Rev. 2016
LAD
Occlusion
Adaptative
Remodeling
and
Inflammation
Ischemia Infarct
Reperfusion
Production of reactive
oxygen species, causing
further tissue damage
Chronic remodeling phase
due to altered mechanical
properties
Heart failure
MI Surgery Death Rate
10-15%
(death within 24 h post-MI)
Blanching of
myocardial tissue
and elevation of ST
segment used to
establish ischemia

7. Branching Patterns
LAD coronary artery ligation
Rat
LMCA – Left main coronary artery LAD – Left anterior descending artery LCX – Left circumflex artery RCA- Right coronary artery
Distally from a long LMCA From septal arteryProximally from a short LMCA
Kainuma S. et al. (2017) PLoS ONE 12(8)
Kumar D et al Coron Artery Dis, 2005
Surgical ligation of the left coronary artery in mice
produces MIs that involve the LV free wall and apex
while sparing the septum.
LAD coronary artery ligation
Mouse

8. ANIMAL MODEL OF HF: ISCHEMIA/REPERFUSION
LAD- Left anterior descending TCC- 2,3,5-Triphenyltetrazolium Chloride
Temporary Ligation (I/R Model)
Species
Rats (SD, ZDF, CD® IGS)
Mice (C57BL/6)
Anesthesia
Nembutal
Isoflurane
Ketamine/Xylazine
Body temperature: 36.5-37.5 °C
ECG: Monitored throughout the experiment
Blood sampling: Biomarkers
Rats: Immediately before ischemia
and throughout reperfusion
Mice: Terminal only (0.7-1.0mL)
Hemodynamics
Systemic Arterial
LV pressure
Pressure-Volume Loop
Infarct Size: India Ink/TTC staining (entire heart used)
LAD Coronary
Artery
Ligation
30 to 60 min
Ischemia
Reperfusion
2hr to 3days

9. I/R Models - Intervention
Prevention
Study
Treatment
Study
Baseline Ischemia Reperfusion
Baseline Ischemia Reperfusion
Terminal Procedure
Intervention

10. Formalin
Fixation
LAD
re-Occluded
Heart
Excised and
Sectioned
Frozen heart
sectioned using Slicer
Matrix (2mm sections)
TTC Staining
Section is incubated
with 1% TTC
for 15min at 37˚C
LV slices
Right ventricle,
apex and base is
discarded
Image
Digitized
AAR: Reddish color
IZ: Pale yellow
Tail vein injection
of India Ink
India Ink/ TTC Staining
LAD
TTC is enzymatically
reduced to a red
formazan product by
dehydrogenases.
Stain intensity
correlates with the
number and functional
activity of
mitochondria.
(Goldlust et al., 1996).
Stain of non-
ischemic health
myocardium.
Ischemic area not
stained.
Five serial LV sections
Image J 1.46r
Software
Both sides of the
slide is scanned
using a flat bed
scanner
LAD- Left anterior descending
TCC- 2,3,5-Triphenyltetrazolium Chloride

11. I/R Model: Temporary Ligation
Zucker Diabetic Fatty Rat
Anesthesia: Nembutal
Body temperature: 36.5 to 37.5 °C
Intubated: Ventilation with room air
Proper ligation of the LAD is confirmed by
observing blanching of myocardial tissue and
increase of ST segment in the ECG.
Ischemia: 30 minutes
Reperfusion: 120 minutes
Infarct size: India Ink/TTC staining
Biomarkers: Troponin I
Bleeding Group: Blood samples obtained at Day -2, 1
hour before anesthesia, immediately before
ischemia and at 1 and 2 hours of reperfusion.
No-Bleeding Group: Blood samples obtained
immediately before ischemia and at 1 and 2 hours of
reperfusion.
LAD- Left anterior descending
TCC- 2,3,5-Triphenyltetrazolium Chloride

12. Test 5 Test 18
Bleeding No Bleeding
AAR: Area at risk (pink + pale yellow areas)
IZ: Infarct zone (pale yellow areas)
Test #/
Animal Tag
Area at risk
( % of LV)
Infarct Size
(% of AAR)
Test 9/Tag 10 58.25 19.06
Test 10/Tag 11 51.10 25.07
Test 13/Tag 14 60.85 54.27
Test 14/Tag 15 59.80 32.43
Test 17/Tag 19 80.33 67.23
Test 18/Tag 20 84.18 51.30
Test 21/Tag 24 82.38 44.12
Test 22/Tag 25 60.02 37.25
Test 25/Tag 29 74.65 44.08
Test 26/Tag 30 61.23 32.16
Test 29/Tag 33 69.95 30.68
Test 30/Tag 34 82.39 38.09
Test 6/ Tag 313 69.85 30.10
Test 7/ Tag 316 71.39 20.60
Test 8/ Tag 315 47.38 36.39
Mean 67.58 37.52
SD 11.72 13.04
No Bleeding
Test #/
Animal Tag
Area at risk
( % of LV)
Infarct Size
(% of AAR)
Test 1/ Tag 303 71.94 8.21
Test 2/ Tag 307 80.26 11.82
Test 3/ Tag 305 54.63 5.74
Test 4 / Tag 311 59.78 4.52
Test 5/ Tag 312 59.78 4.52
Mean 65.28 6.96
SD 10.52 3.11
Bleeding
I/R Model in the Zucker Diabetic Fatty Rat

13. I/R Model in the Zucker Diabetic Fatty Rat: No-Bleeding Group

14. Anesthesia: Isoflurane
Body temperature: 36.5-37.5 °C
Intubated: Ventilation with room air
Proper ligation of the LAD is confirmed by
observing blanching of myocardial tissue and
increase of ST segment in the ECG.
Ischemia: 60 minutes
Reperfusion: 240 minutes
Infarct size: India Ink/TTC staining
Left femoral vein: TA bolus injection
Test #
Area at risk
( % of LV)
Infarct Size
(% of AAR)
Infarct Size
(% of Total LV)
Test #
Area at risk
( % of LV)
Infarct Size
(% of AAR)
Infarct Size
(% of Total LV)
12 39.24 15.21 5.97 17 26.09 44.02 11.48
13 38.89 26.81 10.43 18 31.43 46.20 14.52
14 60.07 54.98 33.03 24 48.63 12.76 6.21
15 30.30 37.92 11.49 Mean 35.38 34.33 10.74
16 27.04 42.83 11.58 SEM 6.80 10.80 2.43
Mean 39.11 35.55 14.50
SEM 5.76 6.80 4.74
Vehicle TA
I/R Model in the Mouse (C57BL/6)

15. 60 minutes Ischemia + 240 minutes Reperfusion
Systemic Arterial Pressure and LV Pressure: Carotid artery (Millar)
Mean±SD
n=3/group
I/R Model in the Mouse (C57BL/6)

16. ANIMAL MODEL OF HF: PERMANENT LIGATION
LAD- Left anterior descending
Species
Rats (SD)
Mice (C57BL/6)
Anesthesia: Isoflurane
Body temperature: 36.5 to 37.5 °C
ECG: Monitored during surgery
Biomarkers
Circulating
Heart tissue (Immunohistochemistry)
Hemodynamics
Echocardiography
Pressure Volume Loop (Terminal)
Telemetry
ECG
Arrythmia Analysis
Infarct Size
Histology (Picrosirius Red staining)
LAD Coronary
Artery
Ligation
Ischemia
Day 1
Infarct
Day 15
Heart Failure

17. Chronic MI Intervention
Prevention
Study
Treatment
Study
Surgery 1 Week 2 Weeks 3 Weeks …4 Weeks 16 Weeks
Surgery 1 Week 2 Weeks 3 Weeks …4 Weeks 16 Weeks
LVEF ≤40%
or ≤55%*
*EF randomized between groups
Intervention
LAD
Occlusion
Permanent
Ischemia
EF- Ejection Fraction

18. Weeks
Telemetry Recording (4 days/week)
-5 15 29 57 85542 8
MI
14 28 56 84 112-113-116 Days
Rats (Sprague Dawley)
Dob- Dobutamine PV Loop- Pressure-Volume Loop Echo- Echocardiography ANP- Atrial Natriuretic Peptide
End Points
Hemodynamic Function: Echocardiography, Systemic Pressure (telemetry), PV Loop (terminal)
Electrocardiography: Telemetry, Arrhythmia Detection
Biomarkers: ANP and Troponin I (Plasma)
Infarct Size: Histology (Picrosirius red staining)
Bleed
Bleed Naïve only
AR Analysis Echo Dob Challenge
PV Loop +
Dob challenge Necropsy
Anesthesia: Isoflurane
Body temperature: 36.5 to 37.5 °C
Intubated: Ventilation with room air
DSI Radiotelemetry transmitter implanted prior to MI
Echocardiography
PV Loop + Dobutamine Challenge (Terminal)
Dobutamine Challenge (conscious): Day 54
Inclusion criteria: EF ≤45%
Naïve Sham MIGroups 1 2 3

19. Anesthesia: Isoflurane
Body Temperature: 36.5 to 37.5 °C
Imaged in B-mode in the parasternal long axis (MS250S -Vevo 2100). Trace of the endocardial area will be obtained at diastole
and systole.
Diastole Diastole
Systole Systole
Day -5
Day -5
Day 15
Day 15
Rat Model of Myocardial Infarction: Echocardiography

20. HR- Heart rate LVAd- Left ventricle diastolic area LVESV- Left ventricle end-systolic volume LVAs- Left ventricle systolic area
EF- Ejection fraction FS- Fractional Shortening LVEDV- Left ventricle end-diastolic volume SV- Stroke volume
CO- Cardiac output BW- Body Weight
HR
(bpm)
LVAd
(mm2)
LVAs
(mm2)
CO
(mL/min)
EF
(%)
FS
(%)
SV
(µL)
LVEDV
(µL)
LVESV
(µL)
-5 227±2 405±23 69±5 31±2 87±8 73±1 28±1 215±27 295±36 79±10
15 292±5 389±26 77±4 35±2 98±10 73±1 26±3 252±25 345±34 93±10
113 401±8 379±40 83±6 39±4 106±18 71±2 25±3 279±30 391±50 112±23
-5 166±12 412±5 58±3 25±3 70±4 74±5 27±6 169±14 229±19 60±13
15 277±18 391±21 72±4 33±4 89±10 72±4 25±4 229±21 318±34 90±19
113 487±74 363±24 91±3 44±2 116±2 71±1 22±1 319±21 449±29 130±11
-5 201±17 413±23 61±5 27±4 76±10 74±4 27±4 184±22 248±32 64±15
15 292±26 379±41 92±7 67±6 77±13 41±2 11±2 202±22 494±56 291±39
113 509±119 375±30 117±12 84±9 109±15 40±4 12±3 294±57 729±120 434±77
Each value represents mean±SD.
Naïve (n=6-7)
Sham (n=4-5)
MI (n=6-7)
Cardiac Hemodynamics of Naïve SD, Sham MI and MI Rats
Echocardiographic Parameters
BW
(g)
Study
Days
Groups
Rat Model of Myocardial Infarction: Echocardiography

21. Each value represents mean±SD
Rat Model of Myocardial Infarction: Echocardiography

22. Each value represents mean±SD
Heart Rate
Mean Arterial Pressure
Rat Model of Myocardial Infarction: Telemetry

23. Each value represents mean±SD
Pulse Pressure
Rat Model of Myocardial Infarction: Telemetry

24. PV Loop on Day 116 Post-MI in SD Rats – Closed-Chest Approach
Anesthesia: Isoflurane
(intubated and ventilation 100% O2)
Body temperature: 36.5 to 37.5°C
Systemic Pressure catheter
Femoral Artery
Pressure-Volume catheter
Right Carotid Artery
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
LVVolume (µL)
250200150100500
LVPressure(mmHg)
0
50
100
150
Baseline Dobutamine- 12.5µg/kg Dobutamine- 25µg/kg Wash-out
Rat Model of Myocardial Infarction: PV Loop

25. As expected, we observed a Dobutamine dose-related increase in heart rate.
There was also a decrease in arterial pressure at the tested doses.
Each value represents mean±SD
PV Loop on Day 116 Post-MI in SD Rats

26. Dobutamine effect
is diminished in MI
rats.
Each value represents
mean±SD
PV Loop on Day 116 Post-MI in SD Rats

27. Each value represents mean±SD
Tissue Weight on Day 116 Post-MI in SD Rats

28. LAD- Left anterior descending
Mice (C57BL/6)
Anesthesia
Isoflurane (intubated)
Intubated
Ventilation with room air
Body temperature
36.5 to 37.5 °C
ECG: Monitored throughout the surgery
Proper ligation of the LAD is confirmed by blanching (at the apex and lateral LV) and increase of ST segment
in the ECG.
Hemodynamic Monitoring: Echocardiography at Day 14
End Points
Hemodynamic Function: Echocardiography
Biomarkers: Cytokines (Plasma)
Infarct Size: Histology (Picrosirius red staining)

29. Echocardiography Day 28 Post-MI in C57BL/6 Mice: PLAX LV trace
Sham – Day 28
Systole
MI – Day 28
Systole

30. Each value represents mean±SD
Vehicle: 0.5% methylcellulose
Echocardiography Day 28 Post-MI in C57BL/6 Mice

31. Each value represents mean±SD
Tissue Weight on Day 28 Post-MI in C57BL/6 Mice
Each value represents mean±SD
Vehicle - 0.5% methylcellulose