Observation of the Systolic Function of Isolated Right Atria from Guinea Pigs

Summary

The present protocol describes an efficient method for screening drugs that enhance myocardial contractility using isolated right atria from guinea pigs.

Abstract

Common chronic heart failure (CHF) is characterized by impaired ventricular filling and/or ejection function, which leads to insatiable cardiac output and increased incidence. The decline in cardiac systolic function is a key factor in the pathogenesis of CHF. Systolic function is simply the filling of oxygenated blood in the left ventricle, followed by the blood being pumped throughout the body during a heartbeat. A weak heart and the inability of the left ventricle to contract appropriately as the heart beats indicate poor systolic function. Many traditional herbs have been suggested to strengthen the systolic function of the heart in patients. However, stable and efficient experimental methods for screening compounds that enhance myocardial contractility are still lacking in the process of ethnic medicine research. Here, taking digoxin as an example, a systematic and standardized protocol is provided for screening compounds that enhance myocardial contractility by using isolated right atria from guinea pigs. The results showed that digoxin could markedly enhance the contractility of the right atrium. This systematic and standardized protocol is intended to serve as a methodological reference for screening the active ingredients of ethnic medicines in the treatment of CHF.

Introduction

Heart failure is caused by myocardial infarction, myocardiopathy, hemodynamic overload, inflammation, and other causes of myocardial injuries, which modify the myocardial anatomy and activity and, ultimately, lead to failure in the ventricular pumping or filling. Palpitations, tiredness, and fluid retention are the main primary clinical symptoms¹. CHF is a chronic heart failure condition that can be maintained, deteriorate, or show decompensation over time, and its incidence and prevalence increase with age². The decline in cardiac systolic function is a key factor in the pathogenesis of CHF³. The current medical treatment for the disease mainly involves the use of antihypertensive drugs such as angiotensin-converting enzyme inhibitors, β-adrenoceptors (which inhibit the excessive activation of the neurohormonal system, namely the sympathetic system and the renin-angiotensin-aldosterone system), or diuretics (which reduce congestion)⁴. However, the clinical signs of heart failure caused by reduced cardiac output and reserve are not often addressed in studies examining the impact of these medical treatments⁵.

Positive inotropic drugs are designed to increase myocardial contractility. Cardiac glycosides, phosphodiesterase inhibitors, and β-adrenergic receptor agonists are used as positive inotropic drugs for treating heart failure. Cardiac glycosides are primarily Digitalis derivatives; an example is, digoxin, which is the most prevalently used Digitalis derivative and is derived from Digitalis lanata (white foxglove)⁶. They selectively bind to Na⁺/K⁺-ATPase on the cell membrane to increase the intracellular calcium concentration and, thus, enhance the cardiac contractility and stroke volume without elevating the oxygen intake, thereby improving cardiac efficiency⁷. Aside from cardiac glycosides, most positive inotropic drugs, such as phosphodiesterase inhibitors and β-adrenergic receptor agonists, increase the heart rate and myocardial oxygen consumption while increasing the calcium load in myocardial cells to enhance the myocardial contractility, which can result in clinically severe arrhythmias and hypotension and, thus, increased mortality⁸. Therefore, the clinical application of these inotropic drugs is limited. In order to avoid complications caused by elevated intracellular calcium levels, it is necessary to develop safer and highly effective inotropic modulators for the treatment of CHF (Figure 1).

In recent decades, many studies have been conducted to generate and analyze compounds that can support the positive inotropic properties of cardiac hemodynamics. Many traditional Chinese medicines (TCM), such as Euodia rutaecarpa (Juss.) Benth., Apocynum venetum L., and Sophora alopecuroides L., among others, can enhance myocardial contractility^9,10,11. Studies have proven that TCM and its active monomers can exert positive inotropic effects through different mechanisms compared to inotropic drugs. For example, liguzinediol, a form of ligustrazine methylated at C₂ and C₅ (one active ingredient of Szechwan Lovage Rhizome), which significantly enhances the contractility of isolated rat hearts by enhancing sarcoplasmic reticulum calcium transients without increasing the heart rate, may have fewer side effects and be a better treatment for CHF¹². Additionally, matrine is an alkaloid extracted from the TCM plant Sophora flavescens Ait. Matrine can inhibit the upregulation of β₃-AR protein expression and diminish eNOS expression in heart failure model rats, thereby enhancing their myocardial contractility¹³. However, in ethnic medicine research, there is a lack of stable and efficient experimental methods for screening compounds that can enhance myocardial contractility.

It is commonly known that, compared to other rodents, guinea pigs have electrophysiology and calcium handling characteristics that are more similar to those of humans¹⁴. On the one hand, the electrocardiogram of guinea pigs is sufficiently similar to that of humans, and their beat-to-beat Ca²⁺ handling is more similar to human physiology than that of rats or mice^15,16. On the other hand, computational models of guinea pig cardiomyocytes have undergone extensive research and include crucial cellular subsystems, including energetics and reactive oxygen species metabolism¹⁷. Therefore, isolated right atria from guinea pigs are widely used to screen compounds that enhance myocardial contractility. Here, we take digoxin as an example to provide a systematic and standardized protocol for screening compounds that enhance myocardial contractility by using isolated right atria from guinea pigs. Therefore, this work provides a methodological reference for screening the active ingredients of ethnic medicines in the treatment of CHF.

Protocol

The experimental protocol was conducted in accordance with the requirements of the Use of Laboratory Animals and Institutional Animal Care and Use Committee at Ningxia Medical University. Male Dunkin-Hartley guinea pigs weighing 300-450 g were used for the present study. The effect of digoxin on contractility was observed in isolated right atria from the guinea pigs (Figure 2).

1. Oxygenation preparation for the isolated right atria of guinea pigs

1. Prepare the experimental instruments, including a biological signal acquisition and processing system, a JH-2 muscle force transducer, a Magnus bath, an L-shaped ventilation hook, thick scissors, a Petri dish, paraffin, etc. (see Table of Materials).
2. Prepare 1,000 mL of Krebs-Henseleit solution (K-H solution) by adding 7.02 g of NaCl (120.0 mM), 2.10 g of NaHCO₃ (25.0 mM), 0.30 g of KCl (4.0 mM), 0.07 g of MgSO₄ (0.6 mM), 0.07 g of NaH₂PO₄ (0.6 mM), 0.28 g of CaCl₂ (2.5 mM), and 1.98 g of glucose (11.0 mM) into 1,000 mL of double-distilled water, and rinse the Magnus bath two to three times (see Table 1 and Table of Materials).
   NOTE: Keep the temperature of the K-H solution at 37 °C ± 1 °C.
3. Prepare 100 mL of low-calcium K-H solution by adding 0.70 g of NaCl (120.0 mM), 0.21 g of NaHCO₃ (25.0 mM), 0.03 g of KCl (4.0 mM), 0.01 g of MgSO₄ (0.6 mM), 0.01 g of NaH₂PO₄ (0.6 mM), 0.01 g of CaCl₂ (0.8 mM), and 0.20 g of glucose (11.0 mM) into 100 mL of double-distilled water (see Table 1 and Table of Materials).
4. Place approximately 20 mL of the 37 °C K-H solution in the operating basin (see Table of Materials).
5. Spread 5 mm of thick paraffin over the bottom of the Petri dish, and then fill the Petri dish with the 37 °C K-H solution (see Table of Materials).
6. Install the L-shaped ventilation hook on the latex tube end of the bladder, put it in the Petri dish, and adjust to 1-2 bubbles/s (see Table of Materials).
   ​NOTE: Slowly adjust the bubbles; if the action is too quick, the oxygen may soon run out.

2. Preparation of isolated right atria from guinea pigs

1. Weigh guinea pigs on a scale (see Table of Materials).
2. Induce anesthesia using an induction box with 5% isoflurane in 100% oxygen, and then switch to a nose cone with 1.5%-3% isoflurane for maintenance (see Table of Materials).
3. Cut the carotid artery with rough scissors, and induce exsanguination before placing it on a plate. Then, using scissors, open the thorax (starting with the xiphoid process and completely separating the sides to expose the heart), and peel off the pericardium.
   1. Hold the heart up with the left hand, use the right hand to cut the heart from the root of the aorta, and quickly place it in the operating basin with the K-H solution. Finally, gently press the ventricle with the hand two to three times, squeeze out the ventricular blood, and place the heart in the Petri dish.
      NOTE: The action should be brisk and completed in 2-5 min, and the temperature must be controlled at 35 °C. The temperature of the K-H solution must be controlled at 37 °C.
4. Fix the tip of the heart to the paraffin-coated Petri dish with a needle while providing oxygen (60 bubbles/min).
5. Identify the right atrium.
   NOTE: In guinea pigs, the atria are separated on their ventral surface by the pulmonary artery and dorsally by the aorta. Both the left and right atria are attached to the ventricles like an "inverted triangle", and the right atrium is slightly smaller than the left atrium and has uneven edges. The myocardium of the right ventricle is thin, and the upper end of the collapse is the right atrium; the left ventricle is more puffed, and the distribution of coronary vessels is rich¹⁸ (Figure 3).
6. Gently lift the edge of the right atrium with ophthalmic forceps, and cut along the atrioventricular junction (see Table of Materials).
   NOTE: Avoid damaging the sinoatrial node, and try to cut more close to the ventricle while cutting along the atrioventricular junction. Automatic rhythmic contraction of the right atrium can be observed in this step.
7. Use 4-0 surgical sutures (see Table of Materials) to ligate the top and bottom of the right atrium, respectively (both ends of the "diagonal line"), with one end looped and the other end left with a long thread end that is also looped.
   ​NOTE: Try to ligate as few tissues as possible when ligating both ends of the "diagonal line" of the atria.

3. Measurement and recording of the systolic function of isolated right atria from guinea pigs

1. Turn on the computer, and enter the biological signal acquisition and processing system (see Table of Materials). Adjust the gain (50 mV), time constant (DC), filter (20 Hz), and scanning speed (1.00 s/div) after determining the connection channel (first channel, tension).
2. Hang one end of the specimen on the L-shaped ventilation hook with the Petri dish and oxygen beside the Magnus bath. Hang the other end of the specimen on the JH-2 muscle force transducer (see Table of Materials).
3. Observe the atrial systolic curve, adjust the preload to 0.5-1.0 g, and wait for it to stabilize (about 30 min).
   NOTE: Change the K-H solution every 20 min. When normal, take the observed curve as the standard, and mark "normal". If the screen is scanned a little fast, this must be slowed down.
4. Administer 0.2 mL of low-calcium K-H solution, and observe for 5 min until the curve no longer declines.
5. Administer 0.2 mL of 5% digoxin (see Table of Materials), observe for 5 min, wash three times, and then return to normal.
   NOTE: Mark the administration. When the effect is obvious, scan the screen faster. When the curve no longer rises, that is, when the contraction amplitude no longer increases, wash three times quickly; otherwise, arrhythmia will occur, which will affect the results of the subsequent drug experiments.
6. Collect the data, and save it to a floppy disk.

Results

A decrease in myocardial contractility causes insufficient cardiac output, which leads to CHF (Figure 1). This protocol allowed the recording of the effects of different drugs on the systolic function of isolated right atria from guinea pigs and then the rapid screening of compounds from ethnic drugs that enhance myocardial contractility. After connecting the right atrium, the JH-2 muscle force transducer, and the biological signal acquisition and processing system in steps, the parameters w...

Discussion

The normal rhythmic activity of the heart requires a suitable physical and chemical environment, as does the activity of isolated right atria. Isolated right atria are isolated from the innervation of the body and the direct influence of systemic humoral factors, meaning changes in the activity of the right atria when changing the drugs they are exposed to can be observed. The fundamental causes of bioelectrical activity in excitable cells are changes in the ion permeability of the cell membrane and the subsequent diffus...

Materials

Name	Company	Catalog Number	Comments
4-0 surgical suture	Yangzhou Fuda Medical Devices Co., Ltd
5% Digoxin (soluble in dimethyl sulfoxide)	TCI Shanghai	D1828	CAS: 20830-75-5; Purity: >96.0%
BL-420N biological signal acquisition and processing system	Chengdu Tai Meng Software Co., Ltd	1700142S
CaCl2	Shanghai yuanye Bio-Technology Co., Ltd	S24110	CAS: 10043-52-4; Purity: 96%
Glucose	Shanghai yuanye Bio-Technology Co., Ltd	S11022	CAS: 50-99-7; Purity: 99%
Isoflurane	RWD Life Science Co., Ltd	R510-22-16
JH-2 muscle force transducer	Institute of Aerospace Medical Engineering, Beijing, China
KCl	Shanghai yuanye Bio-Technology Co., Ltd	S24120	CAS: 7447-40-7; Purity: 99.5%
Magnus bath	Shanghai Future Experimental Equipment Co., Ltd	L046525
MgSO4	Shanghai yuanye Bio-Technology Co., Ltd	S24253	CAS: 7487-88-9; Purity: 98%
NaCl	Shanghai yuanye Bio-Technology Co., Ltd	S24119	CAS: 7647-14-5; Purity: 99.5%
NaH2PO4	Shanghai yuanye Bio-Technology Co., Ltd	S24161	CAS: 7558-80-7; Purity: 99%
NaHCO3	Shanghai yuanye Bio-Technology Co., Ltd	S24153	CAS: 144-55-8; Purity: 99.8%
Operating basin	Guangzhou Telekuan Medical Instrument Co., Ltd		305 mm x 230 mm
Ophthalmic forcep	Suzhou Shuanglu Medical Instrument Co., Ltd
Ophthalmic operating scissor	Suzhou Shuanglu Medical Instrument Co., Ltd
Paraffin	Leica Biosystems	39601095
Petri dish	Corning	430167	100 mm x 20 mm
Rodent anesthesia machine	Shanghai Yuyan Instruments Co., Ltd		ABS type (single channel)
Scale	Shanghai Yueping Scientific Instrument Co., Ltd	YP1002
Surgical plate	Zhengzhou Ketai Experiment Equipment Co., Ltd		21 cm x 31 cm
Tissue scissor	Suzhou Shuanglu Medical Instrument Co., Ltd	SL0023