A Surgical Method for Subvalvular Structure Intervention Through a Transaortic Approach in Normal Bama Pigs

Summary

This study presents a surgical method for subvalvular aortic valve structure intervention using targeted tissue cutting via a transaortic approach in normal Bama pigs.

Abstract

Subvalvular structure abnormalities leading to left ventricular outflow tract (LVOT) alterations and myocardial hypertrophy are characteristic anatomical features of structural heart diseases such as hypertrophic cardiomyopathy (HCM). This study establishes a surgical method for aortic subvalvular structure intervention through a transaortic approach in normal Bama pigs. Animals were obtained from a certified experimental pig supplier in Beijing (Licence No. SCXK (Jing) 2018-0008).This method was validated in three healthy female Bama miniature pigs aged approximately 33-35 weeks, weighing 30-35 kg at the time of surgery. The procedure involves a median sternotomy and an anterior aortic wall incision, performed under cardiopulmonary bypass (CPB) support, hypothermic cardiac arrest, and direct visualization of the LVOT and subvalvular regions. Targeted tissue traction, localization, and cutting are utilized to remodel the subvalvular structure.

The complete surgical process includes anesthesia induction, placement of a central venous catheter and urinary catheter, surgical site disinfection and draping, median sternotomy, pericardial incision, and cardiac mobilization. CPB is established through right atrial drainage and retrograde coronary sinus perfusion. Following an anterior aortic wall incision, the left ventricle is accessed across the aortic valve, and subvalvular tissue intervention is performed under cardiac arrest. The procedure concludes with aortic closure, cardiac resumption of beating, CPB withdrawal, and thoracic closure. Throughout the procedure, hemodynamic stability was maintained, and the animal remained in good condition without intraoperative mortality. After surgery, the cut surfaces were smooth, and the surrounding structures remained intact.

This method demonstrates high reproducibility and procedural controllability, providing a stable surgical intervention model for studying subvalvular structure intervention. It also offers a technical platform for conducting histological analysis, surgical validation, and intervention strategy evaluation.

Introduction

Abnormalities in the subvalvular structures of the left ventricle play a crucial role in various cardiovascular diseases, particularly in patients with hypertrophic cardiomyopathy (HCM), where subvalvular anomalies contribute to left ventricular outflow tract (LVOT) obstruction and interventricular septal hypertrophy¹. These structural changes not only impair ventricular filling and ejection but may also lead to severe complications such as ventricular arrhythmias, sudden cardiac death, and chronic heart failure². Clinically, transaortic surgical resection of left ventricular tissues is often employed in such patients to relieve LVOT obstruction and improve prognosis³. However, only a few studies have systematically investigated the physiological changes and ventricular remodeling processes following this surgical intervention, limiting its application in basic research and translational therapeutic development.

Various animal models have been widely employed to study subvalvular structural changes and intervention strategies. These models have significantly contributed to understanding molecular and tissue-level mechanisms; however, they show notable limitations in simulating clinical cardiac surgical procedures, particularly in achieving precise exposure and targeted tissue cutting of the left ventricle. Problems such as anatomical distortion, insufficient surgical precision, and limited tissue sampling remain^4,5,6,7. These deficiencies hinder the broader application of such models in research on surgical intervention mechanisms, postoperative structural intervention, and biomaterial evaluation.

In recent years, animal models simulating left ventricular subvalvular structure intervention via a transaortic approach have remained extremely limited. Existing studies are primarily restricted to modifications of current surgical techniques, attempting to intervene in localized ventricular tissue structures through minimally invasive pathways or energy-based methods. However, these approaches significantly differ from clinical surgical practices in terms of surgical pathways, intraoperative cardiac conditions, and tissue intervention strategies, making it difficult to accurately reproduce the structural, hemodynamic, reperfusion, and mechanical stress responses induced by surgical interventions^8,9. Therefore, in this study, we established a reproducible large animal surgical model by performing a median sternotomy and an anterior aortic wall incision under cardiopulmonary bypass (CPB) support in normal Bama pigs. This model enables direct exposure of the left ventricular outflow tract and the subvalvular region, followed by controlled tissue cutting to promote subvalvular structure intervention, providing a clinically relevant platform for future research.

Specifically, this model was designed to replicate the procedural pathway and operative environment of clinical transaortic septal myectomy used in patients with hypertrophic obstructive cardiomyopathy (HOCM). It is not intended to recapitulate HOCM pathophysiology or serve as a disease model, but rather to reproduce the operative conditions under which subvalvular intervention is performed.

This model serves as a standardized surgical framework for studying subvalvular structural interventions, evaluating novel cardiac surgical devices, and providing a reproducible training platform for experimental cardiac surgery, including surgical training in transaortic LVOT exposure and controlled subvalvular resection, as well as acute tissue-level and histologic studies following defined structural interventions.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Fuwai Hospital Chinese Academy of Medical Sciences, under approval number 0106-1-20-ZX(X)-21. All experiments were conducted in accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals (National Research Council/Institute of Laboratory Animal Resources). Animals were housed under standard environmental conditions (temperature 20-26 °C, humidity 40-60%, 12 h light/dark cycle) with free access to food and water.

1. Experimental animal preparation

1. Obtain normal adult Bama miniature pigs from a certified experimental pig supplier in Beijing (Licence No. SCXK (Jing) 2018-0008).
   NOTE: This method was validated in three healthy female Bama miniature pigs aged approximately 33-35 weeks, weighing 30-35 kg at the time of surgery.
2. House the animals in a standard barrier environment for at least 7 days prior to surgery for acclimatization, under controlled temperature (20-26 °C), humidity (40-60%), and a 12-h light/dark cycle, with free access to food and water.
3. Perform a comprehensive health assessment 24 h before surgery, including evaluation of mental status, appetite, fecal characteristics, skin integrity, gait, and respiratory condition.
4. Withhold food for 12 h and water for 4 h preoperatively to reduce the risk of aspiration during anesthesia.
5. Complete the body weight measurement and identification registration before surgery, along with recording baseline vital signs.
6. On the day of surgery, transfer the animals to the operating area for intravenous access establishment and anesthesia induction preparation.

2. Preoperative preparation

1. Auricular venous access establishment
   1. Before auricular venous catheterization, anesthesia/sedation was induced by intramuscular administration of ketamine hydrochloride (5–10 mg/kg) combined with Sumianxin II (a compound veterinary anesthetic preparation with xylazine hydrochloride as the main component; 1 mg/kg). After adequate sedation was achieved, establish venous access through the marginal auricular vein as follows:
      1. Apply gentle pressure to the proximal portion of the marginal auricular vein to allow venous filling. Using a 22-G intravenous catheter(22 GA × 1.00 in; 0.8 × 25 mm), insert the needle along the course of the vessel from the distal toward the proximal direction.
      2. Upon observing blood return, withdraw the stylet and advance the catheter fully into the vein. Remove the needle core, connect a three-way stopcock, and inject a small amount of sterile saline to confirm patency.
      3. Confirm that no subcutaneous bulging occurs and that infusion proceeds smoothly, then secure the stopcock with adhesive tape to stabilize the catheter.
2. Anesthesia
   CAUTION: Verify adequate anesthesia depth and loss of reflexes before performing any invasive procedures to prevent stress or pain.
   1. Induce anesthesia by injecting 1% propofol solution via the auricular vein at a dose of 2-4 mg/kg. Once the animal loses standing ability, exhibits decreased consciousness, and collapses, immediately transfer it to the center of the operating table.
   2. Position the animal in the supine position and secure all four limbs to the sides of the operating table using restraint straps. Place soft pads under the back and neck to prevent compression injury.
   3. After confirming unconsciousness, administer a single intravenous bolus of fentanyl (3–5 µg/kg) for peri-incisional analgesia, followed by atracurium (0.2 mg/kg) for muscle relaxation.
   4. Perform endotracheal intubation by exposing the glottis with a laryngoscope. Insert an appropriately sized cuffed endotracheal tube (recommended internal diameter 6.5-7.5 mm) into the trachea and advance it to the thoracic segment to avoid occlusion of the right cranial bronchus.
   5. Connect the tube to a mechanical ventilator. Set the ventilator to a tidal volume of 10-12 mL/kg, a respiratory rate of 12-16 breaths/min, FiO₂ 100%, and an I:E ratio of 1:2. Confirm bilateral lung ventilation before securing the endotracheal tube. After anesthesia induction, apply erythromycin ophthalmic ointment to both eyes. Depending on the duration of surgery, reapply the ointment intraoperatively as needed to protect the ocular surface and prevent corneal drying.
   6. Maintain anesthesia during surgery with inhaled 2% isoflurane.
   7. Continuously monitor electrocardiogram (ECG), blood pressure (BP), oxygen saturation (SpO₂), body temperature (Temp), and the anesthesia machine MAC value.
   8. Maintain body temperature at 37 ± 0.5 °C to reduce metabolic demand and ensure myocardial protection under cardiopulmonary bypass. Maintain SpO₂≥ 95% and keep blood pressure within a stable physiological range.
3. Surgical hand preparation
   1. Perform standard surgical hand antisepsis according to institutional protocol. After handwashing, dry the hands using sterile surgical towels before donning sterile gloves.
4. Central venous catheterization under guidewire guidance
   CAUTION: Maintain strict aseptic technique during catheterization. Handle needles and guidewires carefully to prevent accidental injury.
   1. Select the right external jugular vein for venous access. After anesthesia induction, remove hair from the catheterization site. Scrub the skin 2–4 times with povidone-iodine in concentric circles from the puncture site outward. After disinfection, inject 5 mL of 2% lidocaine subcutaneously to achieve local anesthesia.
   2. Connect the puncture needle to a syringe and insert the needle at an angle of 20-30° along the course of the jugular vein toward the cranial direction.
   3. Advance the needle slowly while aspirating continuously until venous blood return is observed. Insert a guidewire gently into the vessel lumenthrough the needle. Stabilize the guidewire and withdraw the needle carefully. Advance a dilator over the guidewire to expand the puncture tract, then remove the dilator.
   4. Insert a central venous catheter over the guidewire into the vein. Advance the catheter to a depth of approximately 10-15 cm, ensuring that the catheter tip is positioned within the cranial vena cava without entering the right atrium to avoid interference with intraoperative cardiac occlusion.
   5. Remove the guidewire. Confirm patency of each lumen by aspiration. Flush each lumen with heparinized saline (50 IU/mL), and lock the catheter using the same solution to maintain patency.
   6. Secure the catheter with sterile sutures. Apply a transparent sterile dressing over the puncture site. Clearly label the lumen function and catheter insertion depth.
5. Urinary catheterization
   1. After anesthesia induction and positioning, disinfect the perineal area using sterile cotton balls soaked in povidone-iodine solution and allow it to dry naturally. Place a sterile drape over the surrounding area, exposing only the urethral orifice. Use only female pigs to facilitate urethral catheterization.
   2. Under sterile conditions, open a Foley catheter kit. Wear sterile gloves, lubricate the catheter tip, and gently insert it into the urethra. Once urine flow is observed, advance the catheter an additional 1-2 cm. Inject sterile distilled water into the catheter balloon to secure it in place. Apply gentle traction to confirm fixation.
   3. Connect the catheter to a closed drainage bag. Secure the tubing to the tail or the side of the operating table with tape to prevent tension or contamination. Record the time of catheter insertion and procedural details. During surgery, continuously monitor urine output and drainage patency. After urinary catheterization, the anesthesiologist changed gloves and performed hand hygiene before further anesthesia-related procedures.
6. Draping and establishment of the sterile surgical field
   1. After anesthesia induction, hair was removed from the planned median thoracic incision site. After surgical hand antisepsis, the surgeon disinfected the thoracic surgical field 2–4 times with povidone-iodine in concentric circles from the planned incision site outward.
   2. Collaborate with the assistant to drape the surgical field. First, place small sterile drapes and secure them with towel clamps. Then place medium-sized drapes, covering the body except for the operative region.
   3. Ensure that the exposed area extends from the manubrium of the sternum to the xiphoid process, approximately 20-25 cm in length and 10-12 cm in width, including the midline sternum and bilateral costal margins.
   4. After draping, perform rapid hand antisepsis again. Don sterile surgical gowns and gloves with the nurse's assistance. Place a large sterile drape with a fenestration, ensuring that only the incision area is exposed while all other regions remain fully covered. Align the fenestration with the center of the incision and secure the margins to prevent fluid leakage, thereby completing the establishment of the sterile surgical field.

3. Cardiac exposure and mobilization

1. Thoracic cavity exposure
   CAUTION: Protect the operator's hands and eyes during sternotomy. Ensure that the retractor is applied slowly to avoid tissue tearing or bone fracture.
   1. Make a midline incision along the sternum, extending from the suprasternal notch to approximately 2 cm below the xiphoid process, using a surgical scalpel to incise the skin and subcutaneous tissues. Maintain an incision length of approximately 20 cm.
   2. Use an electrocautery device to separate the pectoralis major muscles and the periosteum of the sternum, fully exposing the anterior surface of the sternum.
   3. Gently retract the lateral tissues with the assistance of an assistant to maintain a clear surgical field.
   4. After exposing the sternum, insert a Lebsche knife beneath the xiphoid process and apply upward traction.
   5. Using a small hammer, carefully and evenly tap the proximal end of the Lebsche knife to gradually split the sternum along the midline, proceeding cranially until the thoracic cavity is opened.
   6. Maintain continuous upward traction throughout the process to avoid deviation or injury to the pericardium. After completing the sternal division, place a Finochietto rib retractor and slowly expand the thoracic cavity bilaterally to widen the operative field. Avoid excessive retraction to prevent pleural injury or rib fractures.
2. Cardiac mobilization
   1. Identify the course of the bilateral phrenic nerves running along the lateral surfaces of the pericardium (Figure 1A).
   2. Incise the superior margin of the pericardium and extend the incision parallel to the long axis of the animal, keeping it close to the midline to minimize the risk of nerve injury (Figure 1B).
   3. Perform blunt dissection bilaterally to separate the pericardium from the mediastinal pleura and adjacent connective tissues.
   4. After completing the dissection, expose the heart under direct visualization. Confirm clear identification of key anatomical structures, including the aorta, pulmonary artery, right atrial appendage, cardiac apex, and coronary sinus.
3. Establishment of cardiopulmonary bypass (CPB)
   CAUTION: Ensure that all CPB circuit connections are secure and properly grounded. Handle electrical and perfusion devices carefully to prevent leakage or electric shock.
   1. After achieving adequate cardiac exposure, place a purse-string suture at the base of the right atrial appendage. Make a small incision within the suture loop and insert a venous drainage cannula obliquely downward along the axis of the right atrial appendage to establish the venous return pathway for cardiopulmonary bypass.
   2. Advance the cannula approximately 3-4 cm to ensure that its tip enters the right atrial cavity. Secure the cannula to the atrial appendage using silk sutures to prevent slippage or leakage.
   3. Place a purse-string suture at the entrance of the coronary sinus. Create a small incision within the suture loop and insert a perfusion cannula to serve as the inflow pathway for cardioplegia delivery and myocardial perfusion.
   4. Advance the perfusion cannula approximately 1-2 cm into the coronary sinus. Secure it with silk sutures to ensure stable positioning.
   5. Connect all cannulas to the cardiopulmonary bypass machine. Prime the circuit with a mixture of 800 mL of lactated Ringer's solution, 1,000 mL of succinylated gelatin solution, and 2 mL of heparin. De-air the circuit thoroughly and perform a pressure test to confirm stability.
   6. Continuously monitor perfusion parameters and flow rate throughout the procedure, and maintain activated clotting time (ACT) above 480 s through systemic heparinization.
   7. Activate the venous return pump to initiate partial venous drainage. Gradually open the arterial pump to maintain perfusion pressure at 60-80 mmHg. Confirm that there is no leakage and that circuit flow is unobstructed before initiating full cardiopulmonary bypass.
   8. After achieving stable CPB, slowly infuse cooled high-potassium cardioplegia solution via the coronary sinus. Observe the progressive weakening of cardiac contractions until complete cardiac arrest occurs and the electrocardiogram (ECG) displays a flatline. Record the time of cardiac arrest.
   9. Ensure that complete electromechanical arrest and adequate myocardial cooling are achieved before initiating subvalvular intervention. Begin tissue manipulation only after confirming the absence of ventricular contractions and a stable arrest state.
   10. In this protocol, administer a single-dose cardioplegia sufficient to maintain myocardial arrest throughout the subvalvular intervention period. Re-dosing is not routinely required due to the relatively short aortic cross-clamp duration (approximately 30-40 min).
   11. Apply pre-prepared sterile crushed ice saline slush over the cardiac surface to further reduce myocardial temperature, decrease metabolic demand, and enhance myocardial protection.

4. Left ventricular exposure and subvalvular structure intervention via aortic access

CAUTION: Handle sharp instruments and scissors carefully during aortic incision and tissue cutting. Avoid excessive traction that could damage surrounding structures.

1. After achieving cardioplegic arrest and hypothermic protection, make a transverse incision approximately 1.0-1.5 cm in length on the anterior wall of the ascending aorta, located approximately 1.5-2.0 cm above the aortic valve annulus and oriented perpendicular to the long axis of the aorta.
2. Insert two curved sheet hooks into the aortic incision from the left dorsal and ventral sides, respectively. Gently retract the incision laterally to open the aortic root and expand the surgical field (Figure 2A).
3. Introduce a needle holder carrying a preloaded traction suture into the operative field. Place the suture on the target subvalvular tissue to facilitate intraoperative traction and precise localization.
4. Advance small curved scissors through the aortic incision along the axis of the aorta toward the left ventricular chamber. Use the traction suture to expose the target subvalvular region (Figure 2A).
5. Under direct visualization, orient the scissors horizontally and cut the designated subvalvular structures in a controlled manner (Figure 2B). Remove the resected tissue and hand it to the assistant.
6. Thoroughly irrigate the left ventricular chamber and the aortic lumen with sterile saline to remove any residual tissue debris.
7. Close the aortic incision using continuous absorbable sutures. Begin at one end of the incision and sequentially pass the needle through the intima and media, using evenly spaced stitches to ensure complete, watertight closure. After completing the suturing, irrigate the external surface of the closure site again with sterile saline to clean the area.

5. Cardiac resumption and cardiopulmonary bypass weaning

CAUTION: Confirm full deairing of cardiac chambers and perfusion lines before restoring circulation to prevent air embolism.

1. After completing aortic closure, gradually reduce the coronary sinus perfusion rate and discontinue cardioplegia infusion. Simultaneously, gradually decrease the cardiopulmonary bypass (CPB) pump flow rate to allow progressive recovery of spontaneous cardiac activity.
2. Continuously monitor cardiac rhythm using electrocardiography (ECG) to detect the return of sinus rhythm or the presence of arrhythmias.
3. After successful weaning from CPB, administer protamine according to the total intraoperative heparin dose, using an institutional heparin-to-protamine neutralization ratio of approximately 1:1.5 to reverse systemic heparinization. Reassess ACT approximately 10 min after protamine administration until ACT approaches the preoperative level.
4. If spontaneous cardiac rebeating does not occur within several minutes, apply direct defibrillation by placing defibrillator paddles directly on the cardiac surface. Deliver a single low-energy shock and repeat as necessary until organized electrical activity is restored.
5. After restoration of regular ECG waveforms, assess the apical impulse, coronary pulsation, and overall myocardial contractility.
6. Once cardiac rhythm stabilizes and contractile strength is satisfactory, sequentially remove the coronary sinus perfusion cannula and the right atrial venous drainage cannula. Close each cannulation site with sutures to achieve hemostasis.
7. Disconnect the CPB system. Record the total CPB duration, aortic cross-clamp time, and time to cardiac resumption.
8. Thoroughly irrigate the pericardial cavity with sterile saline and aspirate any residual fluid or blood clots.
9. Before sternal closure, place two closed drainage tubes within the pericardial cavity, positioning one on each side of the heart. Exteriorize the drainage tubes through an intercostal space and connect them to external drainage bottles.
10. Close the sternum using four stainless-steel wires in an interrupted configuration. Subsequently, perform a routine layered closure of the thoracic wall.
    NOTE: After completion of thoracic wall closure, ropivacaine (100 mg) was injected subcutaneously around the wound in divided doses for local analgesia. After surgery, animals were weaned from mechanical ventilation and extubated according to blood gas parameters and recovery of spontaneous respiration, and were then transferred to the monitoring unit. Postoperative analgesia was maintained with butorphanol administered subcutaneously at 4 mg three times daily for at least 3 days. Cefuroxime sodium was administered by intravenous infusion at 1.5 g twice daily for 1 week. Chest drainage tubes were removed according to drainage volume and the presence of pleural fluid. The incision was routinely cared for with povidone-iodine disinfection.

Results

This surgical protocol enables stable exposure and controlled cutting of the subvalvular structures within the left ventricle. During the procedure, the aortic incision was accurately positioned, and entry through the aortic opening allowed clear visualization of the left ventricular outflow tract and the associated subvalvular region. The surgeon localized the cutting area by applying traction on the pre-placed suture and completed targeted subvalvular tissue cutting under direct vision using small, curved scissors. Pos...

Discussion

Subvalvular structure abnormalities of the left ventricle represent critical anatomical changes in patients with hypertrophic cardiomyopathy (HCM), leading to impaired ventricular compliance, elevated diastolic pressure, and left ventricular outflow tract (LVOT) obstruction. These structural abnormalities constitute major pathological bases for clinical cardiac dysfunction and adverse outcomes^10,11 . In this study, we established a surgical method in normal Bama ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Injection	-	-	Used for irrigation and solution dilution
Anesthesia Machine	-	-	Delivers inhaled anesthetics and respiratory support
Anesthetics (Propofol, Fentanyl, Atracurium)	-	-	Used for anesthesia induction and maintenance
Aortic Retractor	SINOVIEW	RT37008-25	Used to retract the aorta and improve visibility
Atraumatic Forceps	SINOVIEW	FC22500-24	Used to grasp tissue while minimizing trauma
Cardioplegia Solution (St. Thomas)	-	-	Used for cardiac arrest and protection
Cardiopulmonary Bypass Machine and Tubing	-	-	Used for maintaining circulation during CPB
Central Venous Catheter Kit	-	-	Establishes central venous access
Endotracheal Intubation Set and Ventilator	-	-	Maintains airway during surgery
Flat Hook Retractor	SINOVIEW	RT37000-00	Used for tissue retraction in thoracic cavity
General Retractor	-	-	Used to retract tissue and expose surgical area
Hemostatic Forceps	-	-	Used to clamp blood vessels or tissue for hemostasis
Heparin Sodium Injection	-	-	Used for anticoagulation
IV Catheter·Radiopaque 22 GA × 1.00 in	BD Angiocath	381123	Used to establish venous access
Microsurgical Instrument Basket	SINOVIEW	90X0003	Used to store and transport microsurgical instruments
Minimally Invasive Curved Scissors	SINOVIEW	SC40230-25	Used in minimally invasive procedures for curved cutting
Minimally Invasive Double-joint Scissors	SINOVIEW	SC55001-29	Used for precise cutting in deep operative fields
Needle Holder	-	-	Used to hold suture needles during stitching
Scalpel Handle and Blade	-	-	Used to incise skin and tissues
Sterile Drapes, Sutures, Gloves, Masks	-	-	Used to maintain a sterile surgical field
Sterilization Box for Precision Instruments	SINOVIEW	90X0401	Used for cleaning and sterilizing surgical instruments
Tissue Forceps (Toothed/Non-toothed)	-	-	Used to grasp tissue
Tissue Scissors	SINOVIEW	SC35101-25SC	Used for general tissue cutting
Triple-joint Microforceps	SINOVIEW	FC17010-301	Used in microsurgery for delicate manipulation
Ultra-sharp Scissors	SINOVIEW	SC35101-23UC	Used for precise tissue dissection
Urinary Catheter and Drainage Bag	-	-	Used for urine drainage and monitoring
Vital Signs Monitor	-	-	Monitors ECG, blood pressure, SpO2, temperature