The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

Podsumowanie

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

Streszczenie

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap – previously cast to match the average size of the rat heart – is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 µL/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

Wprowadzenie

Heart failure (HF) is the leading cause of death in humans worldwide¹. HF often occurs because of myocarditis, ischemic insults to the myocardium, and left ventricular remodeling, leading to the progressive deterioration of cardiac contractile function and patients' quality of life. Although advances in cardiology and cardiac surgery have remarkably lowered HF mortality, they merely serve as transient "delayers" of an inevitably progressive disease process that carries significant morbidity. Therefore, the current lack of effective treatment underscores the need to identify novel molecular targets that can prevent or even reverse HF. This includes changes in the extracellular matrix, uncontrolled cardiac immune response, and interactions between cardiac and non-cardiac cells².

It is important to recognize that the microenvironment that cardiac cells are exposed to directly shapes the immune and regenerative response of the injured heart. In the isolated, saline-perfused heart, CT is generated on the heart surface in the form of small droplets that are derived from the interstitial fluid space (i.e., microenvironment), both under physiological and pathophysiological conditions^3,4,5. Therefore, analysis of the CT (i.e., interstitial fluid) may help to identify factors that regulate cardiac metabolism and contractile function⁶ or influence immune cell functions after migration into the injured heart. Potentially, this may lead to the development of novel therapeutic strategies for the treatment of HF.

The collection of CT from murine hearts is technically challenging. In regular Langendorff-perfused hearts, the exclusive collection of CT is difficult because the mixture of the CT with coronary venous effluent perfusate unpredictably dilutes any concentration of metabolites/enzymes released from the interstitial space. One possible strategy to overcome this limitation is to exclude the venous effluent by cannulating the pulmonary and simultaneously ligating the pulmonary vein⁷. However, this method faces difficulties associated with the cannulation and ligation of the pulmonary artery and vein, causing potential leakage of venous effluent into the cardiac transudate. The concept of using a reverse heart model was first introduced by the group of Kammermeier, who inverted the isolated, perfused heart into an upside-down position and placed a thin latex cap on the epicardial surface to continuously sample CT without the contamination of venous effluent^8,9. Using this procedure, CT was shown to provide a very sensitive measure of the metabolites released from the heart⁹, the capillary transfer of fatty acids⁸, and viral particles¹⁰.

More recently, paracrine factors that may regulate the local immune response and augment cardiac angiogenesis¹¹ have been implicated in the beneficial effects of stem cell-based therapy for heart disease. The analysis of CT in the reversed heart may help to chemically identify these individual paracrine factors. In addition, CT may help to identify the factors involved in the in vivo activation of immune cells in the heart.

The detailed description of CT collection from the heart surface, provided here, is experimentally useful for researchers studying the interplay of immune cells, fibroblasts, endothelial cells, and cardiomyocytes in relation to overall cardiac function. As mentioned above, the interstitial fluid carries the information for cell-to-cell communication within the heart, which can conveniently be assessed by the collection of CT. The detailed technical description, including a video protocol of how to collect CT from the reversed heart, should facilitate the future application of this unique technique.

Protokół

All experiments were approved by the local regulatory agency (LANUV of Nordrhein-Westfalen, Germany) and were performed according to the guidelines of animal use. Animals were fed with a standard chow diet and received tap water ad libitum. All equipment and chemicals necessary to each step of the experiment are available in the Table of Materials.

1. Preparation of the Latex Cap and Intraventricular Balloon

1. Make an aluminum mold using a milling machine that matches the average size of the rat heart (bodyweight of 300-350 g). Polish the mold with superfine (10/0) emery paper.
   NOTE: The detailed metrics of the mold are shown in Figure 1A.
2. Vertically fix the neck of the aluminum mold to the milling machine to prepare the latex cap.
   NOTE: The milling machine causes the mold to slowly rotate. Alternatively, an electric motor can be used.
3. Pour 20 mL of liquid latex (commercially purchased, see the Table of Materials) into a 50 mL glass beaker.
4. Lower the mold until the entire body of the mold is immersed in the latex solution.
5. Slowly lift the mold (5 cm/min) while rotating.
6. Keep rotating the mold for an additional 15 min, until the latex on the surface of the mold is solidified.
7. Add about 1 g of talcum powder to the surface of the mold (already covered by a thin latex film) to prevent damage while detaching.
8. Gently detach with fingers the already dried latex cap from the mold surface; the latex cap is now ready for use (Figure 1B).
9. Connect the outlet of the latex cap to 15-cm silicon tubing (ID = 0.2 mm), used later for the collection of CT.
10. Fill the ventricular latex balloon with water and firmly fix it onto an L-shaped metal cannula connected to a 1 mL, water-filled syringe (Figure 1C).
    NOTE: This will be used to ensure the upright positioning of the heart (see below).
11. Make sure that the balloon is airtight by performing several deflating/inflating tests with the attached 1 mL syringe.
12. Connect the cannula, via a three-way stop, to a pressure transducer for the future measurement of intraventricular developed pressure (Figure 1C).

2. Preparation of Krebs-Henseleit Buffer (KHB) and the Langendorff Perfusion System

1. Set up a Langendorff perfusion system by using either constant-flow (driven by a roller pump) or constant-pressure (generated by static pressure in a glass column) mode.
   NOTE: Details of the Langendorff heart preparation have been described previously¹².
2. Prepare 2 L of a modified KHB (in mM: 116.02 NaCl, 4.63 KCl, 1.10 MgSO₄·7H₂O, 1.21 K₂HPO₄, 2.52 CaCl₂·2H₂O, 24.88 NaHCO₃, 8.30 D-glucose, and 2.0 sodium pyruvate).
   1. Weigh all chemicals but CaCl₂ and dissolve them in 1.8 L of double-distilled water in a 2-L flask.
   2. Bubble the medium with carbogen (95% O₂/5% CO₂) for at least 5 min for equilibration (pH: 7.4) under magnetic stirring.
   3. Add 0.74 g of CaCl₂.2H₂O and bring up the total volume to 2 L with double-distilled water.
   4. Continue stirring and bubbling the medium with carbogen for an additional 5 min.
   5. Filter the KHB through a 0.2 µm filter to eliminate small particles that may obstruct the microcirculation of the heart.
3. Preparation of the Langendorff perfusion system.
   1. Place the filtered KHB into a pre-warmed water bath (38 °C); keep bubbling with carbogen to generate a pressure of 100 cmH₂O inside the KHB reservoir.
   2. Connect the reservoir to the glass column to establish 100 cmH₂O hydrostatic pressure for the Langendorff perfusion with KHB; continue bubbling the KHB inside the column with carbogen.
   3. Adjust the temperature of the warming system so that the temperature at the aortic cannula outlet is 37 °C.
   4. Ensure that the tubing system is bubble-free.
   5. Oxygenate the KHB with carbogen for an additional 5 min, until the PO₂ in the KHB reaches 500-600 mmHg (measured by a blood-gas analyzer).
4. Set up the perfusion system to run either at a constant pressure of 100 cmH₂O or at a constant flow of about 10-20 mL/min using manual switching. Alternatively, use an interchangeable STH pump controller to instantly switch to the perfusion mode.

3. Isolation and Cannulation of the Heart

NOTE: Male Wistar rats with bodyweights of 300-350 g were used so that the sizes of hearts matched the pre-cast latex cap. Rats underwent either a ligation of the left arterial descending (LAD) for 50 min, followed by reperfusion or were sham operated. Details of the methodology for the induction of myocardial infarction (MI) were reported elsewhere¹³. The reversed-heart experiments in the infarct animals were performed 5 days after operation.

1. Anesthetize rats using an isoflurane vaporizer (2% V/V) connected to an animal holding chamber (20 L).
2. Transfer the rats to an operation table (not temperature controlled) after deep anesthesia is reached.
3. Lift the skin and muscle just below the sternum using forceps and cut around the lower margin of the ribs with heavy scissors.
4. Using fine scissors, make a small cut into the diaphragm, at the rib margin. Cut the ribs caudally to make a flap of the entire ventral chest wall.
5. Gently grab the heart with the thumb and index and middle fingers and slowly lift it upwards so that the cardiac vessels become slightly stretched.
6. Excise the heart until the aorta is fully exposed.
7. Place the heart in a 100 mL beaker containing 50 mL of ice-cold KHB (4 °C) and move it to the perfusion apparatus.
8. Immediately mount the heart via the aorta onto a dripping cannula and securely tighten it with a suture (4-0). Avoid air bubbles entering the heart.
9. Apply constant perfusion pressure (100 cmH₂O). Alternatively, a full flow rate (starting with 20 mL/min) can be applied.
   Note: The time from the opening of the thorax to the attachment of the heart to the perfusion cannula should take about 3 min in the hands of an experienced operator.

4. Reversed-heart Model

1. Gently rotate the aortic cannula until the posterior wall of the heart is in en face view.
2. Remove the connective tissue with scissors to expose the opening of the left atrium, making it ready for intraventricular cannulation.
3. Insert the deflated latex balloon attached to a rigid catheter via the left atrium into the left ventricle.
4. Inflate the balloon until it fills the entire ventricular cavity (the inflating volume is pre-marked on the syringe).
5. Invert the heart until it is upside-down, supporting it by the intraventricular balloon catheter.
6. As demonstrated in Figure 1C, mechanically stabilize the inverted heart in an upright position using the intraventricular balloon with a rigid metal catheter.
7. Adjust the position of the heart to avoid excessive twisting of the aortic root.
8. Adjust the diastolic pressure to 3-5 mmHg (measured by the intra-ventricular balloon; see Figure 1C).
9. Observe the epicardial surface of the heart and ensure that small droplets form.
10. Place the latex cap onto the surface of the heart by gently pushing it to cover the entire heart using the fingers.
11. Make sure that the latex cap covers most of the ventricular surface.
12. Remove the air bubbles, if any, inside the cap and the tubing by gently sucking with a 1-mL syringe.
13. Adjust the distal opening of the CT-letting tubing to 10 cm below the horizontal level of the heart.
    NOTE: This procedure ensures slight sucking by negative hydrostatic pressure.
14. Collect drops of CT in a 1.5 mL collection tube placed in ice mixed 1:1 with NaCl. Collect about 0.15-1.5 mL of CT.
    NOTE: The ice/NaCl mixture stabilizes the temperature in the collection tube to below zero (about -4 °C).
    NOTE: The sampling time depends on the experimental purpose. The flow rate of the CT is about 27 ± 20 µL/ min in sham-operated animals (n = 3) and 100 ± 47 µL/min for the coronary-ligated animals (n = 11).
15. Weigh and snap-freeze CT samples in liquid nitrogen and store them at -80 °C for later measurements.

5. Analysis of the CT

1. Use the CT fluid for the analysis of metabolites, depending on the scientific question.
   NOTE: The data shown in Figure 2 and Figure 3 were collected from a constant-pressure perfusion (100 cmH₂O), and about 0.15-1.5 mL of CT fluid was collected within a period of 10 min. This time and volume were sufficient for the proteomic (minimum: 50 µL; Figure 2)¹⁴ and HPLC (minimum: 20 µL; Figure 3)¹⁵ analysis of various purines.

Wyniki

The reversed-heart model enables the collection of cardiac interstitial transudate in an isolated, retro-perfused rat heart (Figure 1A-C). When perfused at a constant pressure of 100 cmH₂O, the rate of interstitial fluid formation ranged between 17 and 147 µL/min, amounting to 0.1-1% of the coronary venous effluent in the isolated heart.

The protein content of the CT...

Dyskusje

The reversed-heart model is based on the well-established Langendorff heart perfusion technique¹² and is performed by simply inverting the heart into an upside-down position and holding this position using a rigid intra-ventricular balloon catheter. In such a way, cardiac interstitial transudate can be physically separated from coronary venous effluent perfusate, dripping by gravity from the base of the heart⁹. The CT can be continuously collected by means of a thin and fle...

Materiały

Name	Company	Catalog Number	Comments
Latex Solution	ProChemie	Z-Latex LA-TZ	http://kautschukgesellschaft.de/%E2%80%A8z-latex-la-tz
Aluminum Mold	Home made	-	Reverse heart model
Universal Ovens	Memmert	UNB 400	Reverse heart model
Latex Balloon	Hugo Sachs	Size 4	Reverse heart model
Milling Machine	Proxxon	MF70	Reverse heart model
Sodium Chloride	Sigma	SZBD0810V	Chemicals
Sodium Hydrogen Carbonate	Roth	68852	Chemicals
Potassium Chloride	Merck	49361	Chemicals
Magnesium Sulphate Heptahydrate	Merck	58861	Chemicals
Potassium Dihydrogen Phosphate	Merck	48731	Chemicals
D(+)-Glucose Anhydrous	Merck	83371	Chemicals
Calcium Chloride Dihydrate	Fluka	21097	Chemicals
Balance	VWR	SE 1202	Weighing chemicals
Double Distilled Water	Millpore	-	Disolving chemicals
Medical Pressure Transducer	Gold	-	Langendorff apparatus
Medical Flow Probe	Transonic	3PXN	Langendorff apparatus
Heating Circulating Bath	Haake	B3 ; DC1	Langendorff apparatus
Laboratory and Vaccum Tubing	Tygon	R-3603	Langendorff apparatus
Animal Research Flowmeters	Transonic	T206	Langendorff apparatus
PowerLab Data Acquisition Device	AD Instruments	Chart 7.1	Langendorff apparatus
LabChart Data Acquisition Software	AD Instruments	Chart 7.1	Langendorff apparatus
Peristaltic Pump	Glison	MINIPULS 3	Langendorff apparatus
Glass Water Column	home made	-	Langendorff apparatus
Water Bath Protective Agent	VWR	462-7000	Langendorff apparatus
Sterile Disposable Filters (0.2 µm)	Thermo Scientific	595-4520	Langendorff apparatus
Blood gas analyzers	Radiometer	ABL90 FLEX PLUS	Gas analyzer
70% ethanol	VWR	UN1170	Cleaning  tubings
100% ethanol	Merck	64-17-5	Cleaning tubings
Wistar Rats	Janvier	-	Animals
Stainless Scissors	AESCULAP	BC702R	Surgical Instruments
Stainless Scissors	AESCULAP	BC257R	Surgical Instruments
Big Forceps	AESCULAP	-	Surgical Instruments
8m/m Stainless Forceps	F.S.T	11052-10	Surgical Instruments
superfine (10/0) emery paper	3M	051111-11694	Reverse heart model