An Introduction to Isolated Langendorff Heart: Experimental Considerations and Best Practices

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.

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5. Historical Background

6. Principles of Langendorff Perfusion

7. Perfusion Systems

8. Constant Flow vs Constant Pressure

9. 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. Key Proteomic Processes and Techniques 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 10 M r 120 kD 70 30 10 50 20 90 Baseline Normoxic 30 mins Normoxic 75 mins Normoxic Normoxic Perfusion Effectively Washes out Blood Normoxic Perfusion Effectively Washes out Blood

23. 60 mins Isch / 20 mins Rep pH 3 10 pH 3 10 pH 3 10 M r 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 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. Lessons from the Langendorff: Heart Lessons from the Langendorff: Heart

27. Lessons from the Langendorff: Perfusion Lessons from the Langendorff: Perfusion

28. References and Readings\ 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