Langendorff Heart

The concept of the isolated perfused heart system, was introduced by Oscar Langendorff more than a century ago and is now a predominant technique in pharmacological and physiological research. The technique allows the examination of cardiac contractile strength (inotropic effects), heart rate (chronotropic effects) and vascular effects without the neuronal and hormonal complications of an intact animal model.

In the Langendorff preparation, the aorta is cannulated and the heart is perfused in a retrograde (reverse) fashion, usually with a nutrient rich, oxygenated solution. The pressure of the solution causes the aortic valve to shut and the perfusate is then forced into the ostium and into the coronary vessels. This allows the heart to beat for several hours.
 
The technique originally required elevated reservoirs to provide a constant, gravity supplied pressure but the technique and equipment have evolved to include both constant pressure and constant flow models in both recirculating and non-recirculating modes.

ADInstruments offer a range of complete systems as well as instruments, transducers and accessories for recording and analyzing parameters from both the Langendorff isolated heart in a number of species.
 

For adequate perfusion of the heart a suitably sized cannula for insertion into the aorta is provided. Once the heart is cannulated and successfully beating, there are several measurements that can be made from the Langendorff preparation.

Perfusion Pressure/Coronary Flow Rate
Langendorff studies are normally carried out in either ‘constant flow’ or ‘constant pressure’ modes. To maintain these different modes of operation, ADInstruments offer the STH Pump controller which uses a simple feedback system to allow the switching from one to the other with the push of a button. This eliminates the need for elevated reservoirs.

• Constant Flow Mode - perfusate is pushed through the heart at a constant rate and the perfusion pressure is measured to give a representation of the coronary resistance
• Constant Pressure Mode - the desired pressure is maintained and the resulting fluctuations in the rate of coronary flow are measured

Left Ventricular Pressure
Left Ventricular pressure is typically measured using a fluid filled balloon-tipped catheter connected to a pressure transducer. The balloon is placed into the left ventricle and inflated until a resting pressure of approximately 3-10mmHg is achieved.

The latex balloons & Teflon catheters can be supplied by ADInstruments for hearts of rats or larger animals, however they can also be made quite easily.  Commonly used materials are food wrap (cling film) and condom tips. See the Intraventricular Pressure Measurement in a Langendorff Preparation (92 KB) technique note for more details.

Mikro-tip catheters can be used for the pressure measurements, but these would need to be placed inside a fluid filled balloon also in order to register the pressure changes.  Note: Pressure-Volume measurements cannot be carried out on the Langendorff preparation due to the nature of the perfusion.  Whilst the coronary arteries are perfused causing the heart to contract, the ventricles are not filling as they would in the normal physiological set-up.

Contractile Force
A simple method that can be used to measure the contractile force of the heart, is to connect the apex to a force transducer and bridge amp via a pulley system.  Tension is applied to the thread attaching the heart to the transducer and changes in contractile force can be monitored.

Electrical Activity
The cardiac electrical activity of a langendorff preparation can be measured using a bioamplifier and suitable electrodes.  Typically, one electrode is connected to the apex of the heart and one to the atria.  The ground electrode can be connected to the aortic cannula. Alternatively, with a suitable electrode, monophasic action potentials can be measured form single cardiac cells.

Temperature
It is important that a stable temperature is maintained in the isolated heart.  The temperature can be measured with a t-type temperature probe and pod. The probe can be inserted into the perfusate flow or into the heart.

Pacing
The heart may be paced using an external stimulator with a stimulus that exceeds the natural cardiac pacemaker rate, after the sinoatrial node is crushed or the right atrium excised. Pacing voltage is determined as a set percentage (normally 110-150%) above the voltage required to capture (pace) the heart and usually should not have to exceed 3-5V with a duration of 0.1 to 1 msec. The PowerLab data acquisition system has an analog output that can be used to control the frequency of the stimulator pulses.

pH & O2 concentration
It is important to maintain the correct pH and a suitable oxygen concentration of the perfusate solution. These can be measured if required using a suitable pH electrode and pH amplifier and a suitable dip type or flow through oxygen electrode.  To measure the oxygen consumption, O₂ electrodes can be placed in the inflow and in the effluent and the difference in concentration calculated.

LabChart
LabChart software (for Windows and Macintosh) combines the familiar simplicity of a traditional strip chart recorder with the powerful analysis features of a digital acquisition system. LabChart software and a PowerLab data acquisition unit provide data integrity, easy selection of hardware settings, powerful online and offline analysis, procedure automation, seamless extraction of experimental data and flexible display options. Acquisition and analysis capabilities can be further increased with LabChart Extensions and LabChart Modules. LabChart Modules are available as part of LabChart Pro and LabChart Extensions are free for download from the website for existing LabChart users.

Using LabChart, the following raw data can be recorded from a Langendorff experiment:

• Perfusion Flow Rate
• Perfusion pressure
• Left Ventricular Pressure (LVP)
• ECG
• Temperature

The following calculated measurements can be made using LabChart functionality:

• Heart Rate (cyclic rate of LVP or ECG)
• Left Ventricular Systolic & Diastolic Pressure (cyclic Maximum and Minimum of LVP)
• dP/dT (1st Derivative of LVP)

LabChart Extensions inlcude:

• Cardiac Axis LabChart Extension: Automate thecalculation of frontal plane ECGs and vector cardiograms and display ofthe instantaneous cardiac vector.
• SAECG(signal averaged ECG) LabChart Extension (MAC only): Calculates theaverage cycle of ECG signals and automatically identifies specificationwaveforms and cardiac indices.
• PeakParameters LabChart Extension: Determines a number of parameters for anindividual peak and is ideal for analyzing cardiac action potentials.

Blood Pressure Module (Windows)
The MLS370/6 Blood Pressure Module for Windows automatically detects, analyzes and reports cardiovascular parameters from arterial or ventricular pressure signals.


The settings dialog allows the user to select ventricular pressure for analysis and the Classifier View allows for easy selection of pressure waveforms for further analyses. Pressure cycles that are contaminated by artifact, have abnormal cycle heights or cycle durations (frequency) can be excluded from analysis using the classifier.

The Analysis View displays pressure cycles as beat-by-beat or as the average of a specified number of cycles. Ventricular parameters such as EDP, Max dP/dt, Min dP/dt, Min and Max Pressure are labeled. These measurements along with other calculated parameters are logged in the Table View for easy exporting.

Features and benefits include:

• Suitable for analysis of pressure signals from humans as well as large and small animals
• Pressure signals can be analyzed in real time during acquisition
• The BP Classifier makes detection and exclusion of atypical waveforms easy
• Parameters can be displayed as continuous data on separate channels
• Values are logged to the Table View
• Averages any number of pressure waveforms

Calculated arterial parameters include:

• Systolic, Diastolic and Mean Pressure
• Pulse Pressure
• Dichrotic Notch Pressure
• Ejection and Non-ejection duration
• Cycle Duration
• Heart Rate

Calculated ventricular parameters include:

• Maximum Pressure
• Maximum dP/dt
• Isovolumic Relaxation
• Minimum Pressure
• Minimum dP/dt
• Mean Pressure
• End Diastolic Pressure (EDP)
• Maximum-Minimum Pressure
• Contractility Index

ECG Analysis Module (Windows)
The MLS360/6 ECG Analysis Module provides a comprehensive set of tools that automatically detects and reports values of ECG recordings. The software can be used with ECG recordings taken from humans and many species of animals from pigs through to mice.

It provides

• Real-time analysis and data extraction of ECG parameters
• PQRST amplitudes
• Time intervals such as RR, PR, JT, QT and QTc
• Analysis of ECG parameters in real-time or offline
• Automated detection and averaging of ECG cycles
• Automated tabulation and data extraction of ECG parameters
• Automated real-time or offline ECG Plots
• Graphical QT vs RR, QT vs Time & RR vs Time plots
• Waterfall plot

Heart Rate Variability (HRV) Module (Windows or Macintosh)
The MLS310 HRV Module provides a comprehensive set of tools for the analysis and display of variation in the interval between heartbeats in human and animal electrocardiogram recordings.

The HRV module provides:

• Detects and analyzes R waves & RR interval variation in ECG real-time or offline recordings
• Includes or excludes ectopic beats from analysis
• Adds R waves or remove short artifacts from analysis
• Provides data export features
  
• Automated HRV Analysis Windows
• Poincaré Plot, Tachogram & Spectrum
• Period Histogram & Delta NN Histogram

Dose Response Module (Windows)
The MLS390/6 Dose Response Module provides easy analysis of dose response type data (response to stimulation by chemical, electrical or physical agonists) recorded in LabChart from various studies including:

• Muscle contraction
• Enzyme activity
• Hormone secretion
• Heart rate
• Blood pressure
• Membrane potential

This module provides:

• Real-time or offline analysis
• Automated or manual modes of analysis
• Fast analysis of raw data to dose response parameters
• Fast comment detection and conversion to dose response markers
• Easy options for calculating different response parameters
• Instant single or multiple dose response curves (Hill-curves) generation
• Instant calculation of EC50 and Hill slopes  
• Export options to other software applications for further analysis

GLP and 21 CFR Part 11
For those researchers working within a laboratory requiring GLP and 21 CFR Part 11 compliance the GLP Client and GLP Server are available for use with LabChart (Windows only) and PowerLab data acquisition systems. For more information, visit the Good Laboratory Practice application page or contact your nearest ADInstruments representative.

ADInstruments provides  a number of versatile hardware solutions for Langendorff experiments.

Compact Langendorff System
The ML870B2 Langendorff System (Constant Pressure or Flow) is a complete system that includes the components for all of the main measurements required from a Langendorff  experiment. It is only suitable for use with hearts from small animals such as rats and mice. The inclusion of the STH pump controller means the apparatus is compact without compromising perfusion pressure and flow measurements.  This system includes:

• ML870 PowerLab 8/30 with LabChart and Scope software
• MLS250 LabChart Pro software
• ML176 Langendorff Apparatus and Thermostat Controller
• ML172 Minipuls-3 Peristaltic Pump
• ML175 STH Pump Controller
• ML221 Bridge Amps (2x)
• ML136 Animal Bio Amp
• MLA1210 Spring Clip Electrodes (Set of 3)
• ML312 T-Type Pod
• MLT844 Physiological Pressure Transducers (2x)
• MLT1401 T-Type Implantable Thermocouple Probe (IT-18)
• Transducer Accessory Kit includes
  
• Connection tubing (Luer Male Lock to Female, 300mm)
• Luer 4-Way Male Lock Stopcock
• Luer Male Lock plug
• Luer Male Lock Slip Connector and Transducer Bracket

Modular Langendorff Systems
Alternatively, more traditional modular glassware systems are also available for Langendorff experiments that include:

ML870B1-V Langendorff System for Mice (Constant Pressure)
A complete system for studying the Langendorff isolated mouse heart using a recirculating, constant pressure system.

ML870B3-V Langendorff System for Rats (Constant Flow)
A complete system for studying the Langendorff isolated rat heart using a non-recirculating, constant flow system.

ML870B4-V Langendorff System for Rats (Constant Pressure)
A complete system for studying the Langendorff isolated rat heart using a non-recirculating, constant pressure system.

Additionally, all of the Working heart systems are can be used additionally in the Langendorff mode, making them an ideal choice if you are unsure which perfusion method to use or if both methods are required,

Note: The Pump supplied with these systems is not suitable for use with the STH pump controller.

Aprotinin impairs coronary endothelial function and down-regulates endothelial NOS in rat coronary microvascular endothelial cells

S Ulker, P P McKeown and U Bayraktutan, Cardiovascular Research, 830-837, 2002

Hearts were excised from anticoagulated, anaesthetised male Sprague–Dawley rats (350–450 g; 100 U i.v. heparin and 100 mg / kg i.p. pentobarbitone sodium) and immersed in ice-cold Krebs–Henseleit solution of the following composition (mM): NaCl 118.0, KCl 4.5, KH2PO4 1.4, MgSO4 1.2, NaHCO3 25, CaCl2 1.4 and glucose 11; pH 7.4. Hearts were initially perfused retrogradely through the aorta by means of a Langendorff heart set-up at a constant flow with Krebs–Henseleit solution maintained at 37şC and gassed with 95% O and 5% CO . The flow rate was determined according to animal weight using the formula: flow (ml/min)=x^0.56 x 7.43 (x is the heart weight), heart weight (mg)=0.0027y+0.6 (y is the body weight). Coronary perfusion pressure was monitored continuously as an index of coronary microvascular tone with a pressure transducer (SensoNor Sp 844, Norway) connected to a side arm of the aortic cannula. Since total flow through the coronary vascular system changes depending on the cardiac contractile activity, left ventricular pressure, as an index of cardiac contractility, was also measured with a fluid-filled latex balloon inserted into the left ventricle and connected to a second pressure transducer. Balloon volume was adjusted to obtain an end-diastolic pressure of 10 mmHg. All variables were recorded continuously on a computer through a data-acquisition system (Chart v4.1, Powerlab / 4SP, ADInstruments, UK). Heart rate, left ventricular developed pressure (LVDP, the difference between systolic and diastolic left ventricular pressures) and the positive and negative differentiated pressures (+dP/dt and -dP/dt) were monitored simultaneously as computed inputs derived from left ventricular pressure……….The heart was allowed to equilibrate for at least 30 min in order to obtain stable cardiac parameters.

Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule

J E Clark, P Naughton, S Shurey, C J Green, T R Johnson, B E Mann, R Foresti and R Motterlini, Circulation Research, e2-e8, 2003

Isolated hearts from male Lewis rats (300 to 350 g) were perfused according to the Langendorff technique at constant flow. Coronary perfusion pressure (CPP), end-diastolic pressure, left ventricular developed pressure (LVDP), heart rate (HR), maximal contraction (+dP/dt), and relaxation (-dP/dt) rates were continuously recorded throughout the period of perfusion using PowerLab (AD Instruments). Isolated hearts were equilibrated for 20 minutes, made globally ischemic for 30 minutes, and then reperfused for 60 minutes. Krebs buffer was collected for 10 minutes from the pulmonary artery before the ischemic event and in the last 10 minutes of reperfusion for creatine kinase (CK) analysis.

PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury

T Nagoshi, T Matsui, T Aoyama, A Leri, P Anversa, L Li, W Ogawa, F del Monte, J K Gwathmey, L Grazette, B Hemmings, D A Kass, H C Champion and A Rosenzweig, Journal of Clinical Investigation, 2128-2138, 2005

Mice were heparinized (1,000 IU/kg, i.p.) and anesthetized (pentobarbital, 60 mg/kg, i.p.) and hearts excised and immersed in icecold perfusion buffer. Aortae were cannulated and retrograde-perfused at a constant pressure (80 mmHg) with modified Krebs-Henseleit buffer (11 mM glucose, 118 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 0.5 mM EDTA, equilibrated with 95% O2/5% CO2 at a pH of 7.4) at 37°C. A water-filled balloon catheter was introduced into the LV for recording of pressure and heart rate (PowerLab; ADInstruments). The balloon was inflated until a stable LVEDP less than 10 mmHg was obtained. The temperature was maintained at 37°C with a water jacket. We monitored coronary flow rate by collecting coronary sinus effluent. No difference in contractile performance was seen between male and female mice, so data from both sexes were combined in all studies.

Protection against endotoxemia-induced contractile dysfunction in mice with cardiac-specific expression of slow skeletal troponin I

J Layland, A C Cave, C Warren, D J Grieve, E Sparks, J C Kentish, R J Solaro and A M Shah, FASEB Journal, , 2005

Male transgenic mice hearts were retrogradely perfused with Krebs-Henseleit buffer (KHB) containing (mM): 118 NaCl, 3.8 KCl, 1.18 KH2PO4, 25 NaHCO3, 1.19 MgSO4, 1.25 CaCl2, 10 glucose, 5.0 Napyruvate, and bubbled with 95% O2-5% CO2 at 37°C. Coronary flow was adjusted to achieve a coronary perfusion pressure of 75 mmHg, which was thereafter kept constant using a feedback device. Hearts were paced at 588 bpm via the atria. Isovolumic left ventricular pressure (LVP) was measured using a water-filled polythene balloon inserted into the LV. The balloon was inflated in 5 µl increments, and LVP was measured at each volume. Data were sampled at 1 kHz via a PowerLab module (AD Instruments, Hastings, UK) running Chart software (version 4.1.2). Measurements of maximum left ventricular pressure (max LVP), LV end diastolic pressure (LVEDP), and LV developed pressure (LVDP=max LVP-LVEDP) were derived from the resulting LVP trace. The Chart software also displayed the derivative of LVP on-line, from which the maximum rates of LVP rise (LV dP/dtmax) and decline (LV dP/dtmin) were measured.

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in the heart

M Frederich, L Zhang and J A Balschi, American Journal of Physiology: Heart and Circulatory Physiology, 2412-2421, 2005

Hearts of male Sprague-Dawley rats (body wt, 280–320 g) were isolated and perfused in the isovolumic Langendorff model. Krebs-Henseleit buffer (KH) perfusate contained (in mM) 118 NaCl, 5.9 KCl, 1.2 MgSO4, 25 NaHCO3, 1.75 CaCl2, 0.5 sodium EDTA, and 10 D-glucose and was equilibrated with 95% O2-5% CO2 with a resultant pH of 7.4. A Stratham P23dB pressure transducer connected to the left ventricle balloon measured the left ventricular pressure and heart rate (HR). Hearts were electrically paced using a Grass stimulator (Grass; Quincy, MA). Left ventricular pressures were recorded with a MacLab system (ADInstruments; Colorado Springs, CO).