In this webinar, Dr. Melanie White, Heart Foundation Future Leader Fellow from the University of Sydney, provides a useful introduction to isolated heart studies. Key topics covered during this webinar include: - Understanding the core principles of isolated Langendorff perfusion - Key methodological considerations for excision, cannulation and perfusion of the heart - Experimental design: when to use constant flow vs. constant perfusion, animal models (species, sex, age) and choice of anesthesia - How to set up your hardware to ensure your experiments are trouble-free - Tips for data analysis: using a baseline period, defining exclusion criteria and evaluating functional output - Applications of Langendorff perfusion, from myocardial ischemia to diabetic cardiomyopathy

1. An Introduction to Isolated
Langendorff Heart: Experimental
Considerations and Best Practices
Melanie White, PhD
Heart Foundation Future
Leader Fellow
The University of Sydney
School of Medicine

2. An Introduction to Isolated
Langendorff Heart: Experimental
Considerations and Best Practices
Dr. Melanie White discusses basic isolated Langendorff
heart principles, key experimental design considerations,
core technology requirements and best practice tips to
support consistency and validation of your research.

3. InsideScientific is an online
educational environment
designed for life science
researchers. Our goal is to aid
in the sharing and distribution
of scientific information
regarding innovative
technologies, protocols,
research tools and laboratory
services

4. To access webinar content,
Q&A reports, FAQ
documents, and information
on lab workshops,
subscribe to our mail list

5. Work by Carl
Ludwig and Elias
Cyon on isolated
Frog hearts
•Frog hearts were
popular because of a
single ventricle and
no coronary
circulatory system
Oscar
Langendorff
isolated larger
mammalian
hearts
•Defibrinated
blood was used
from the
respective
species
Aortic
cannulation
necessary for
resuscitation
• Coronary
circulation is
the defining
factor for
physiological
function of the
heart
Isolated
working
heart (Neely
& Morgan)
• Implemented
the use of a
modified K-H
bicarb. buffer
with 95% O2
and 5% CO2
Historical Background

6. Coronary circulation
is essential for
physiological contractility
Oxygenated perfusion
solution delivered via the
canular provides
retrograde perfusion
Perfusate options
are designed to mimic
plasma content
Heart can be maintained
using constant pressure
or constant flow
Can be used for precise
induction of numerous
cardiac pathologies
System is free of influence from
other organs, systemic
circulation, humoral factors and
autonomic innervation
Principles of Langendorff Perfusion

7. Crystalloid perfusion
• NaCl, KCl, MgSO4, KH2PO4,
NaHCO3 CaCl2
• Glucose is used as the
primarily carbon source
• Pyruvate can be
supplemented, fatty acids
tend to be insoluble
• Under physiological
conditions the heart will
efficiently extract most
metabolic carbon sources
• Low oncotic pressure and
limited oxygen carriage
Whole blood perfusion
• Donor animal is also required
to simulate the buffer
reservoir
• Requires recirculation
• Risk of haemolysis and
humoral effects
Erythrocyte perfusion
• Bovine origin
• Combined with crystalloid
buffer and albumin
• Physiological osmolality and
oncotic pressure
Perfusion Systems

8. Peristaltic pump inserted between buffer reservoir and cannular allows for control of flow or pressure
• Can be calibrated to remove the need for flow meters
Constant flow experiments will measure perfusion pressure changes
• Allows for consistency across experiments
• Unable to provide autoregulatory mechanisms
Constant perfusion pressure achieved using negative feedback circuit using perfusion pressure
measured controls the pump speed
• Allows autoregulation of vascular tone
• Blood perfused hearts will perfuse at a smaller volume
Usual flow rates (range of 8-12ml/min/g) – significantly higher than physiological levels
Constant Flow vs Constant Pressure

9. Smaller rodents: mouse, rats
Larger rodents: guinea pig
Small mammals: rabbits
Species to be used
No significant differences in normoxic function
Females can have smaller infarct zones
Sex differences
LVDP changes in normoxic perfusion and post-Ischemia (+ sex effects)
Flow rates (constant pressure)
Age differences
Inhalants
Injection – intraperitoneal or intravenous
Choice of
anesthesia
Important Considerations

10. • Deep anaesthesia is required
• Check for auditory and pain reflex
• Barbiturates are used frequently
• Narrow therapeutic range to define deep sedation vs
cardiorespiratory suppression
• Heparin can be delivered to reduce clotting
• Thoracotomy is used to expose the heart and great vessels
Excision of the Heart
Click Here to
Watch the
Webinar

11. Cannulation of the Heart
• Aorta must be dissected above the root but below the aortic
arch
• Within the aortic sinuses are the openings of the left and right
coronary circulation
• Heart is placed in cold cardioplegic solution to limit the effects
of hypoxia
• Cannulating the aorta is easily the most challenging component
and will depend on the size of the animal
• Once ligated on the cannular, the flow can be increased and
perfusion commenced
Cannulation of the Heart

12. • Baseline periods run for 15–20 mins
• This allows for washout of toxic
metabolites from the heart prior to
initiating protocols
• Allows for observation of individual
heart functionality
• Can apply exclusion criterion for
perfusion studies
• Once ligated on the cannular, the
flow can be increased and perfusion
commenced
The Benefit of a Baseline Period

13. Monitoring
Functional Output
• Perfusion pressure
• Aortic pressure
• Flow rate
• Heart rate
• Diastolic pressure
• Systolic pressure
• ECG/EKG
• Calculate left ventricular
developed pressure (LVDP)
• Contractile force developed in
the LV
• Systolic – diastolic pressures
• Calculate rate pressure product
• LVDP x heart rate (bpm)

14. Exclusion Criteria
• End baseline RPP
<26,000
• Flow rate <10ml/min
• Numerous arrythmias
lasting >15sec

15. Exclusion Criteria
• End baseline RPP
<26,000
• Flow rate <10ml/min
• Numerous arrythmias
lasting >15sec

16. Limitations and Caveats
• Decline in contractile and chronotropic
function over time
• 5-10% per hour
• Typically bradycardic by comparison to
in vivo heart rate
• Mouse: 380-420 bpm vs 580–600 bpm
• Rat: 250-320 bpm vs 350–400 bpm
• Clinical viability
• This included the clinical viability
of model used
• Recycling perfusate solutions can
lead to conditions similar to
metabolic acidosis

17. • Pentobarbitol i.p.
• Loss of pain reflex
• Injection of heparin directly into renal
artery
• Heart is plunged into ice cold saline
• Time from opening of diaphragm to
hanging is <120sec
• Low flow is maintained while aorta is
secured
• Flow is increased gradually to
approximately 13-15ml/min
• Constant pressure
• 20 mins baseline
• Balloon is set with an afterload between
10-15 mmHg
Langendorff in Action

18. • Our aim is to understand cellular adaptations to:
– Ischemia / reperfusion injury (I/R)
– Type 2 Diabetes (T2D)
• To develop hypothesis in an unbiased way, we
use proteomics
• The proteome is the protein component of the
genome – what is translated
• Proteomics can sample across the cell, taking a
snapshot of the cellular response to changing
extracellular stimuli
• Utility becomes limited beyond 4 orders of
magnitude
The White Lab: Cardiometabolic Proteomics

19. • Detergents, denaturants, reductants and alkylating agentsSample solubilisation
• MW separation (SDS-PAGE)
• pI separation (IEF)
Gel based separation
• Trypsin
• Lys-C/Lys-N
Enzymatic digestion of proteins
• Size exclusion
• Hydrophobicity (C18) / Hydrophilicity (HILIC)
• Strong cation/anion exchange
Liquid Chromatography
• SILAC
• TMT/iTRAQ
• Label-free approaches
Quantitative approaches
• LC-MS/MS
• MALDI-MS/MS
Mass Spectrometry
Key Proteomic Processes and Techniques

20. Myocardial Stunning
Acute myocardial infarction
Biomarker discovery
Reactive oxygen species scavenging
Ischemic time course
Reperfusion time course
Pharmacological interventions
Ischemic Pre-Conditioning
Ischemic Post-Conditioning
Diabetic cardiomyopathy
Applications
of Langendorff
Perfusion
We use Langendorff Perfusion systems
to investigate:

21. Collecting the
Perfusate
• Proteomics can be limited
by samples with broad
dynamic ranges (heart and
blood)
• Aim: Define a better
biomarker of Ischemia /
Reperfusion injury
• Approach: Collect perfusate
after ejection from the
heart as a rich source of
coronary biomarkers
• Methods: Langendorff
perfusion and proteomic
techniques
Release of Tissue-specific Proteins into Coronary Perfusate as a Model for
Biomarker Discovery in Myocardial Ischemia/Reperfusion Injury
Stuart J. Cordwell,Alistair V. G. Edwards,Kiersten A. Liddy,Lia Moshkanbaryans,Nestor Solis, Benjamin L. Parker, Andy S. C.
Yong, Clement Wong, Leonard Kritharides, Brett D. Hambly,Melanie Y. White
Abstract
Diagnosis of acute coronary syndromes is based on
protein biomarkers, such as the cardiac troponins
(cTnI/cTnT) and creatine kinase (CK-
MB) that are released into the circulation. Biomarker discovery is focused on identifying very low
abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of
the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit
model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion.
Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief,
reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins
were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry
(MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during
irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis
coupled to liquid chromatography and tandem mass spectrometry; geLC–MS) and gel-free (LC–MS/MS)
methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified
proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to
examples currently under investigation in large cohort studies (heart-type fatty acid binding protein;
FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich
protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified
Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies
indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the
presence of obfuscating plasma proteins may identify candidates for further study in humans.

22. pH
3 10
pH
3 10
pH
3 10Mr
120 kD
70
30
10
50
20
90
Baseline
Normoxic
30 mins
Normoxic
75 mins
Normoxic
Normoxic Perfusion Effectively Washes out Blood

23. 60 mins Isch /
20 mins Rep
pH
3 10
pH
3 10
pH
3 10Mr
120 kD
70
30
10
50
20
90
Baseline
Normoxic
15 mins Isch /
60 mins Rep
Collection of Post Ischemic Perfusate Introduces Cardiomyocyte
Proteins to Profile

24. CSRP3 is Effective for Defining I/R Injury – Clinical Specimens

25. • Underlying issues of the animal will influence the ability of the
heart to recover from the excision process
– This includes handling stress
• Heparin should be used
• Don’t swap anesthetics
• Physiological contractility is retained for longer if the heart
is submerged in the organ bath
• Ensure you can observe the end of the canular through the aorta
– This ensures you haven’t perforated the aortic valve
• Home made balloons can be tricky to make, but worth the effort
• All steps of the process take practice!
Lessons we have learnt using Langendorff Perfusion
Click Here to learn more
about ADI’s solutions for
Langendorff Heart Perfusion

26. Condition of the animal
will influence the
function of the heart
Heparin is important
Contractility is
maintained when the
heart is submerged
Ensure you can see the
end of the canular
through the aorta
Homemade balloons
are tricky, but worth
the effort
Practice, practice,
practice
Lessons from the Langendorff: Heart

27. Lessons from the Langendorff: Perfusion
Keep dedicated
glassware
Water source is
important
Everything needs to be
filtered repeatedly
Air bubbles are akin to
inducing an ischemic
insult
Ensure appropriate
cleaning protocols
To maintain flow rates,
release tubing from
peristaltic pump (when
not in use)

28. • Zimmer H-G. 1998
• Sumeray M.S. and Yellon D.M. 1998
• Sutherland F.J. and Hearse D.J. 2000
• Headrick J.P. et al 2001
• Sutherland F.J. et al 2003
• Johnson M.S. et al 2006
• Skyped-Spring M. et al 2007
• Reichelt M.E. et al 2009
• Bell R.M. et al 2011
• Liao R. et al 2012
• Motayagheni N. 2017
References and Readings

29. • White Lab
• Desmond Li
• Lauren Smith
• Meaghan Morris
• Harriet Wadsworth
• Nina Hartcher
• Prajwal Thapa
• Patrick McNamara
Dr Melanie White was supported by a Future Leader Fellow (102009)
from the National Heart Foundation, Australia
Acknowledgements
• Cordwell Lab
• Stuart Cordwell
• Alexander Rookyard
• Alistair Edwards
• Benjamin Parker
• Jana Paulech
• Kiersten Liddy
• Angela Connolly

30. Melanie White, PhD
Heart Foundation Future
Leader Fellow
The University of Sydney
School of Medicine
Thank You
To learn more about ADInstrument’s products and solutions for Langendorff Isolated
Heart Perfusion, please visit: www.adinstruments.com