This work was supported by the National Science Foundation of China (no. 82200309) and the Innovation Project of Distinguished Medical Team in Ningbo (no. 2022020405)

All of the procedures presented here conformed to the principles outlined in the Declaration of Helsinki and were approved by the Animal Welfare and Human Studies Committee at Shanghai Children&#39;s Medical Center (SCMC-LAWEC-2023-003). C57BL/6 mice pups (P7, males, 3-4 g) were used for the present study. The animals were obtained from a commercial source (see Table of Materials). The mice pups and their nursing mothers (pups:mothers = 6:1 in a single cage) were kept under specific-pathogen-free laboratory conditions under a 12 h light and dark cycle at 22 &#177; 2 &#176;C with free access to water and a nutritional diet. The pups were randomized into two groups: a VO group and a sham-operated (sham) group.
1. Equipment and surgical tool preparation
NOTE: The commercial details of all the materials/equipment are listed in the Table of Materials.
<ol>
	<li>Ensure that the following types of equipment are ready and properly functioning: operating table (foam plastic panel), inhalational anesthesia machine, microscope with vertical illumination and a built-in camera, ultrasound device with a 24 MHz transducer, and thermostatic heating platform.</li>
	<li>Sterilize the surgical instruments (i.e., a micro-needle holder, fine-tip forceps, and round-handled Vannas spring scissors).</li>
	<li>Assemble the following consumables: 11-0 and 9-0 surgical suture needles (taper-point) with thread, tape strips, 5 mL syringe needles, 2-0 silk (surgical fixation), sterile cotton swabs, and ultrasound gel.</li>
	<li>Ensure the following reagents are present: Betadine, 70% ethanol, normal sterile saline, isoflurane, acetaminophen, ophthalmic ointment, and hair removal cream.</li>
</ol>
2. Surgical procedure
NOTE: The fistula surgery procedure was modified according to the previously described method11. Figure 1 shows a schematic diagram of the AVF operation in postnatal mice.
<ol>
	<li>Anesthesia and restraint
	<ol>
		<li>Place the mice pups into an anesthesia-induction box supplied with 2% isoflurane/oxygen for 2 min&#160;with the flow set to 1 L/min. Administer acetaminophen (0.1 ml PO of 80 mg/2.5 ml) using a TB syringe.</li>
		<li>Place the pups in a supine position on the operating table with nasal inhalation of 1.5% isoflurane with a 0.8 L/min flow to maintain anesthesia. Adjust the pup&#39;s position by tying the legs to the fixed syringe needles. Apply ophthalmic ointment to the pups&#39; eyes to prevent corneal desiccation.</li>
		<li>Pinch the anesthetized pup&#39;s tail to check its pain responsiveness; no obvious body movements indicate adequate anesthesia.</li>
	</ol>
	</li>
	<li>Fistula surgery
	<ol>
		<li>Disinfect the skin with three alternating scrubs of betadine and 70% ethanol, and then drape the surgical site. Cut the abdominal wall and peritoneum from the lower abdomen to the subxiphoid to fully expose the peritoneal cavity, taking care not to injure the abdominal organs. Drip normal sterile saline to moisten externalized organs.</li>
		<li>Gently pull the gastrointestinal tract and bladder away from the surgical site using cotton swabs to visualize the vertical abdominal aorta (AA) and inferior vena cava (IVC) under the retroperitoneum. Rotate the operating table 90&#176; counterclockwise and adjust the microscope magnification to visualize the two horizontal vessels clearly.</li>
		<li>Puncture the fistula from the AA into the IVC in an oblique direction 1 cm distal to the renal artery with an 11-0 suture needle (diameter = 0.07 mm). Verify successful fistula creation based on the swelling and mixing of venous and arterial blood in the IVC.</li>
		<li>Next, rapidly compress the bleeding point using an appropriate force applied with dry cotton swabs for 15 s. Replace the stomach, intestines, and bladder in the abdominal cavity as soon as possible to promote hemostatic compression.</li>
		<li>Suture the abdominal wall and peritoneum with a blanket stitch using a 9-0 suture thread. Discontinue anesthesia and provide the pups with 100% oxygen for 1 min.</li>
	</ol>
	</li>
	<li>Anesthesia resuscitation
	<ol>
		<li>Place the pups on a 38 &#176;C heating platform. After a complete awakening with vitality, return the pups to their nursing mother. The entire procedure lasts approximately 15 min. 
		&#8203;NOTE: In the present study, the sham group undergoes the same procedure except for the puncturing step.</li>
	</ol>
	</li>
</ol>
3. Ultrasound confirmation of fistula
NOTE: The general operation of the ultrasound device was identical to previous reports18,19.
<ol>
	<li>Confirmation of fistula by abdominal ultrasound
	<ol>
		<li>After induction of anesthesia (step 2.1.1), fix the mice with tape strips in the supine position on the warm platform. Then, connect the mice to an electrocardiogram (ECG) monitor with ultrasound gel. Maintain anesthesia using 1.5% isoflurane at a 0.8 L/min flow.</li>
		<li>Prepare the chest and abdominal skin using hair removal cream. After a few seconds, remove the cream with a warm water-soaked cotton tip. Place the transducer (24 MHz) on the midabdominal line and rotate the transducer marker to the head of the mice.</li>
		<li>Move the platform down to the left or right side of the mice and use B-mode and color Doppler mode to visualize the long-axis view of the vessels and blood signals18,19. Measure the blood flow velocity of the AA, IVC, and fistula to confirm AVF patency through pulsed wave Doppler mode. 
		NOTE: Successful fistula creation on the ultrasound was indicated by a turbulent flow signal visible between the AA and IVC (Figure 2C). Doppler blood flow velocity at the AVF site was significantly elevated compared with a relatively lower systolic velocity in the AA (Figure 2A,C). Moreover, in contrast with normal flow patterns in the IVC (Figure 2B), the pulsatile waveform of IVC blood flow proximal to the AVF also confirmed the successful creation of the fistula (Figure 2D).</li>
	</ol>
	</li>
	<li>Confirmation of VO by echocardiography
	<ol>
		<li>Move the tail-end part of the platform downward, place the transducer (24 MHz) on the chest, and rotate the transducer marker to the right shoulder of the mice. Visualize the modified parasternal long-axis view of the pulmonary artery (PA) using B-mode and color Doppler mode.</li>
		<li>Using pulsed wave Doppler mode, measure blood flow signals in the PA, including the velocity time integral (PA-VTI), diameter of the PA valve (PAD), pulmonary arterial acceleration time (PAT), and RV ejection time (RVET) (Figure 2E,F and Figure 3A,B).</li>
		<li>Measure the ultrasound parameters from the mean of three consecutive measurements. Calculate the RV stroke volume (RVSV, mL) and RV systolic pressure (RVSP, mmHg) using the following formulas20: 
		RVSV [mL] =1/4&#160;&#215;&#160;&#960;D2 &#215; VTIPA 
		RVSP [mmHg]&#160;= -83.7 &#215; PAT/RVET - index + 63.7 
		NOTE: Considering the ultrasound measurement bias, an increase of &#62;15% in the RVSV or VTIPA in VO mice compared to mice in the sham group was considered VO in the RV (Figure 2E,F).</li>
	</ol>
	</li>
</ol>

Modified Arteriovenous Fistula Surgery to Establish the RV Volume Overload Mouse Model

<ol>
	<li>Sanz, J., Sanchez-Quintana, D., Bossone, E., Bogaard, H. J., Naeije, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy,+function,+and+dysfunction+of+the+right+ventricle:+JACC+state-of-the-art+review.">Anatomy, function, and dysfunction of the right ventricle: JACC state-of-the-art review.</a> Journal of the American College of Cardiology. 73 (12), 1463-1482 (2019).</li><li>Alonso-Gonzalez, R., Dimopoulos, K., Ho, S., Oliver, J. M., Gatzoulis, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+right+heart+and+pulmonary+circulation+(IX).+The+right+heart+in+adults+with+congenital+heart+disease.">The right heart and pulmonary circulation (IX). The right heart in adults with congenital heart disease.</a> Revista Espa&#241;ola de Cardiolog&#237;a. 63 (9), 1070-1086 (2010).</li><li>Kovacs, A., Lakatos, B., Tokodi, M., Merkely, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+mechanical+pattern+in+health+and+disease:+beyond+longitudinal+shortening.">Right ventricular mechanical pattern in health and disease: beyond longitudinal shortening.</a> Heart Failure Reviews. 24 (4), 511-520 (2019).</li><li>Ye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+blood+oxygen+saturation+during+postnatal+human+cardiomyocyte+cell+cycle+activities.">Role of blood oxygen saturation during postnatal human cardiomyocyte cell cycle activities.</a> JACC: Basic to Translational Science. 5 (5), 447-460 (2020).</li><li>Ye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+overload+greatly+promotes+neonatal+right+ventricular+cardiomyocyte+proliferation:+a+new+model+for+the+study+of+heart+regeneration.">Pressure overload greatly promotes neonatal right ventricular cardiomyocyte proliferation: a new model for the study of heart regeneration.</a> Journal of the American Heart Association. 9 (11), e015574 (2020).</li><li>Geraets, I. M. E., Glatz, J. F. C., Luiken, J., Nabben, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+role+of+membrane+substrate+transporters+on+the+metabolic+alterations+in+the+pressure-overloaded+heart.">Pivotal role of membrane substrate transporters on the metabolic alterations in the pressure-overloaded heart.</a> Cardiovascular Research. 115 (6), 1000-1012 (2019).</li><li>Burns, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+mechanistic+and+therapeutic+targets+for+pediatric+heart+failure:+report+from+a+National+Heart,+Lung,+and+Blood+Institute+working+group.">New mechanistic and therapeutic targets for pediatric heart failure: report from a National Heart, Lung, and Blood Institute working group.</a> Circulation. 130 (1), 79-86 (2014).</li><li>Shaddy, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carvedilol+for+children+and+adolescents+with+heart+failure:+a+randomized+controlled+trial.">Carvedilol for children and adolescents with heart failure: a randomized controlled trial.</a> Journal of the American Medical Association. 298 (10), 1171-1179 (2007).</li><li>Flaim, S. F., Minteer, W. J., Nellis, S. H., Clark, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+arteriovenous+shunt:+evaluation+of+a+model+for+heart+failure+in+rat.">Chronic arteriovenous shunt: evaluation of a model for heart failure in rat.</a> American Journal of Physiology. 236 (5), H698-H704 (1979).</li><li>Liu, Z., Hilbelink, D. R., Crockett, W. B., Gerdes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+changes+in+hemodynamics+and+cardiac+myocyte+size+in+rats+with+aortocaval+fistulas.+1.+Developing+and+established+hypertrophy.">Regional changes in hemodynamics and cardiac myocyte size in rats with aortocaval fistulas. 1. Developing and established hypertrophy.</a> Circulation Research. 69 (1), 52-58 (1991).</li><li>Scheuermann-Freestone, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+congestive+heart+failure+in+the+mouse+due+to+chronic+volume+overload.">A new model of congestive heart failure in the mouse due to chronic volume overload.</a> European Journal of Heart Failure. 3 (5), 535-543 (2001).</li><li>Du, Y., Plante, E., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+evaluation+of+cardiac+myocyte+hypertrophy+and+hyperplasia+in+male+rats+secondary+to+chronic+volume+overload.">Temporal evaluation of cardiac myocyte hypertrophy and hyperplasia in male rats secondary to chronic volume overload.</a> The American Journal of Pathology. 177 (3), 1155-1163 (2010).</li><li>Wu, J., Luo, X., Huang, Y., He, Y., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+right-ventricle+functional+characteristics+of+a+swine+carotid+artery-jugular+vein+shunt+model+of+pulmonary+arterial+hypertension:+An+18-month+experimental+study.">Hemodynamics and right-ventricle functional characteristics of a swine carotid artery-jugular vein shunt model of pulmonary arterial hypertension: An 18-month experimental study.</a> Experimental Biology and Medicine. 240 (10), 1362-1372 (2015).</li><li>Sun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+right+ventricular+developmental+track+changed+by+volume+overload.">Postnatal right ventricular developmental track changed by volume overload.</a> Journal of the American Heart Association. 10 (16), e020854 (2021).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+maturation+during+postnatal+right+ventricular+development+switches+to+heart-contraction+regulation+due+to+volume+overload.">Metabolic maturation during postnatal right ventricular development switches to heart-contraction regulation due to volume overload.</a> Journal of Cardiology. 79 (1), 110-120 (2022).</li><li>Zhou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulated+developmental+processes+in+the+postnatal+right+ventricle+under+the+influence+of+a+volume+overload.">Downregulated developmental processes in the postnatal right ventricle under the influence of a volume overload.</a> Cell Death Discovery. 7 (1), 208 (2021).</li><li>Cui, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+overload+initiates+an+immune+response+in+the+right+ventricle+at+the+neonatal+stage.">Volume overload initiates an immune response in the right ventricle at the neonatal stage.</a> Frontiers in Cardiovascular Medicine. 8, 772336 (2021).</li><li>Cheng, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+right+ventricular+structure+and+function+in+mouse+model+of+pulmonary+artery+constriction+by+transthoracic+echocardiography.">Assessment of right ventricular structure and function in mouse model of pulmonary artery constriction by transthoracic echocardiography.</a> Journal of Visualized Experiments. (84), e51041 (2014).</li><li>Sawada, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+imaging+of+the+thoracic+and+abdominal+aorta+in+mice+to+determine+aneurysm+dimensions.">Ultrasound imaging of the thoracic and abdominal aorta in mice to determine aneurysm dimensions.</a> Journal of Visualized Experiments. (145), e59013 (2019).</li><li>Thibault, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+assessment+of+murine+pulmonary+arterial+pressure:+validation+and+application+to+models+of+pulmonary+hypertension.">Noninvasive assessment of murine pulmonary arterial pressure: validation and application to models of pulmonary hypertension.</a> Circulation: Cardiovascular Imaging. 3 (2), 157-163 (2010).</li><li>Mori, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+dynamic+three-dimensional+digital+color+doppler+method+for+quantification+of+pulmonary+regurgitation:+validation+study+in+an+animal+model.">A new dynamic three-dimensional digital color doppler method for quantification of pulmonary regurgitation: validation study in an animal model.</a> Journal of the American College of Cardiology. 40 (6), 1179-1185 (2002).</li><li>Bossers, G. P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+load-induced+right+ventricular+dysfunction+in+animal+models:+insights+in+a+translational+gap+in+congenital+heart+disease.">Volume load-induced right ventricular dysfunction in animal models: insights in a translational gap in congenital heart disease.</a> European Journal of Heart Failure. 20 (4), 808-812 (2018).</li><li>Yamamoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+aortocaval+fistula+recapitulates+human+arteriovenous+fistula+maturation.">The mouse aortocaval fistula recapitulates human arteriovenous fistula maturation.</a> American Journal of Physiology. Heart and Circulatory Physiology. 305 (12), H1718-H1725 (2013).</li><li>Jouannic, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+a+systemic+arteriovenous+fistula+on+the+pulmonary+arterial+blood+pressure+in+the+fetal+sheep.">The effect of a systemic arteriovenous fistula on the pulmonary arterial blood pressure in the fetal sheep.</a> Prenatal Diagnosis. 22 (1), 48-51 (2002).</li><li>Jouannic, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+arteriovenous+fistula+leads+to+pulmonary+artery+remodeling+and+abnormal+vasoreactivity+in+the+fetal+lamb.">Systemic arteriovenous fistula leads to pulmonary artery remodeling and abnormal vasoreactivity in the fetal lamb.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 285 (3), L701-L709 (2003).</li><li>Patel, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+assessment+of+right+ventricular+afterload+in+preterm+infants:+maturational+patterns+of+pulmonary+artery+acceleration+time+over+the+first+year+of+age+and+implications+for+pulmonary+hypertension.">Echocardiographic assessment of right ventricular afterload in preterm infants: maturational patterns of pulmonary artery acceleration time over the first year of age and implications for pulmonary hypertension.</a> Journal of the American Society of Echocardiography. 32 (7), 884-894 (2019).</li><li>Habash, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+values+of+the+pulmonary+artery+acceleration+time+(PAAT)+and+the+right+ventricular+ejection+time+(RVET)+in+children+and+adolescents+and+the+impact+of+the+PAAT/RVET-index+in+the+assessment+of+pulmonary+hypertension.">Normal values of the pulmonary artery acceleration time (PAAT) and the right ventricular ejection time (RVET) in children and adolescents and the impact of the PAAT/RVET-index in the assessment of pulmonary hypertension.</a> The International Journal of Cardiovascular Imaging. 35 (2), 295-306 (2019).</li><li>Arkles, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+of+the+right+ventricular+Doppler+envelope+predicts+hemodynamics+and+right+heart+function+in+pulmonary+hypertension.">Shape of the right ventricular Doppler envelope predicts hemodynamics and right heart function in pulmonary hypertension.</a> American Journal of Respiratory and Critical Care Medicine. 183 (2), 268-276 (2011).</li></ol>

There are no conflicts of interest to declare.

Previously, the classic RV VO model was created using valve regurgitation21; however, compared to AVF, open-heart valve surgery may require more sophisticated techniques and may be associated with significantly higher mortality, particularly in postnatal mice. As animal studies have shown that the same effect of VO has been achieved by AVF22, modified abdominal fistula surgery with less trauma was used in this study.
Certain factors were consider...

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease (CHD), which leads to pathological myocardial remodeling and a poor long-term prognosis1,2,3. An in-depth understanding of RV remodeling and related early targeted interventions is essential for a good outcome in children with CHD. There are several differences in the molecular structures, physiological functions, and responses to stimuli in the hearts of adults and children1,4,5,6. For example, under the influence of pressure overload, cardiomyocyte proliferation is the main response in neonatal hearts, whereas fibrosis occurs in adult hearts5,6. In addition, many effective drugs in treating heart failure in adults have no therapeutic effect on heart failure in children, and may even cause further damage7,8. Therefore, conclusions drawn from adult animals cannot be directly applied to young animals.
The arteriovenous fistula (AVF) model has been used to induce chronic heart VO and corresponding cardiac dysfunction for decades in adult animals of different species9,10,11,12,13. However, little is known about the model in postnatal mice. In our previous studies, a VO postnatal mouse model was successfully generated by the creation of an abdominal AVF. The changed RV developmental track in the postnatal heart was also demonstrated14,15,16,17.
To explore the underlying modified surgical process and characteristics of the present model, a detailed protocol is presented; the model is evaluated for 3 months in this study.

<table><tbody><tr><td>70% Ethanol</td><td>Tiandz,Chia</td><td></td><td></td></tr><tr><td>ACETAMINOPHEN Oral Solution</td><td>VistaPharm, Inc. Largo, FL 33771, USA</td><td>NDC 66689-054-01</td><td></td></tr><tr><td>Anesthesia machine</td><td>RWD Life Science,China</td><td>R550IP</td><td></td></tr><tr><td>Anesthesia mask</td><td>RWD Life Science,China</td><td>68680</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Xipu&amp;rsquo;er-bikai Experimental Animal Co., Ltd (Shanghai, China)</td><td></td><td></td></tr><tr><td>Hair removal cream</td><td>Veet, France</td><td>VT-200</td><td></td></tr><tr><td>Hematoxylin and eosin Kit&amp;nbsp;</td><td>Beyotime biotech&amp;nbsp;</td><td>C0105M&amp;nbsp;</td><td></td></tr><tr><td>Isoflurane</td><td>RWD Life Science,China</td><td>R510-22-10</td><td></td></tr><tr><td>Microscope</td><td>&amp;nbsp;Yuyan Instruments, China</td><td>SM-301</td><td></td></tr><tr><td>Surgical suture needles</td><td>NINGBO MEDICAL NEEDLE CO.,LTD, China</td><td></td><td></td></tr><tr><td>Thermostatic heating platform</td><td>Qingdao Juchuang Environmental Protection Group Co., Ltd, China</td><td></td><td></td></tr><tr><td>Ultrasound device</td><td>FUJIFILM VisualSonics, Inc.</td><td>Vevo 2100</td><td>Image modes includes B-Mode, Color Doppler Mode and Pulsed Wave Doppler Mode</td></tr><tr><td>Ultrasound gel</td><td>Parker Laboratories,United States</td><td>REF 01-08</td><td></td></tr><tr><td>Ultrasound transducer</td><td>FUJIFILM VisualSonics, Inc.</td><td>MS 400</td><td></td></tr></tbody></table>

establishment and confirmation of a postnatal right ventricular volume overload mouse model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Survival rate and AVF patency within 3 months 
A total of 30 (75%) mice in the VO group and 19 (95%) mice in the sham group survived the AVF surgery (Figure 4A). In the VO group, eight mice died within 1 day after surgery due to excessive bleeding (n = 5) or cannibalization (n = 3), whereas two mice died of unknown causes at 1 month.
Of the surviving VO mice (n = 30), ultrasound confirmed the successful establishment of fistulas in 21 mice po...

Watch this Scientific Journal Video about Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model at JoVE.com

Author Spotlight: Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV ...

establishment-confirmation-postnatal-right-ventricular-volume

Research

JoVE Journal

Medicine

1.0K Views.  Shanghai Jiaotong University. Ons onderzoeksgebied is de aangeboren hartafwijking en hartregeneratie. We proberen te begrijpen hoe volume- of drukoverbelasting de postnatale ontwikkeling van hart en longen beïnvloedt, inclusief proliferatie en rijping van cardiomyocyten. We hebben verschillende soorten postnatale muis- of rattenmodellen van aangeboren hartafwijkingen ontwikkeld.Bijvoorbeeld ventriculaire overbelasting, drukoverbelasting en de verminderde pulmonale doorbloeding. Deze modellen zijn fascinerend en zo...

Video: Auteur in de schijnwerpers: Oprichting en bevestiging van een postnataal rechterventrikel volume-overbelastingsmuismodel  

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. ...

A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. Mechanistic studies to improve our&#160;understanding of the underlying pathophysiology responsible for the development of premature TV failure are lacking. Due to the inability to conduct these studies in humans, an animal model is required. In this manuscript, we describe the protocols for a novel chronic recovery infant piglet heart model for the study of changes in the TV when placed under combined volume and pressure stress. In this model, volume loading of the right ventricle&#160;and the TV is achieved through the disruption of the pulmonary valve. Then pressure loading is accomplished through the placement of a pulmonary artery band. The success of this model is assessed at four weeks post intervention surgery through echocardiography, intracardiac pressure measurement, and pathologic examination of the heart specimens.

A novel recovery piglet heart model with combined pressure and volume overload on the right ventricle is described for the study of tricuspid valve function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Biology

Creating a Murine Model of RV Hypertrophy and Failure by Pulmonary Trunk Banding

A Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure by Pulmonary Trunk Banding

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary diseases. The pathogenesis of RV failure is complex and remains inadequately understood. To identify new therapeutic strategies for the treatment of RV failure, robust and reproducible animal models are essential. Models of pulmonary trunk banding (PTB) have gained popularity, as RV function can be assessed independently of changes in the pulmonary vasculature.
In this paper, we present a murine model of RV pressure overload induced by PTB in 5-week-old mice. The model can be used to induce different degrees of RV pathology, ranging from mild RV hypertrophy to decompensated RV failure. Detailed protocols for intubation, PTB surgery, and phenotyping by echocardiography are included in the paper. Furthermore, instructions for customizing instruments for intubation and PTB surgery are given, enabling fast and inexpensive reproduction of the PTB model.
Titanium ligating clips were used to constrict the pulmonary trunk, ensuring a highly reproducible and operator-independent degree of pulmonary trunk constriction. The severity of PTB was graded by using different inner ligating clip diameters (mild: 450 &#181;m and severe: 250 &#181;m). This resulted in RV pathology ranging from hypertrophy with preserved RV function to decompensated RV failure with reduced cardiac output and extracardiac manifestations. RV function was assessed by echocardiography at 1 week and 3 weeks after surgery. Examples of echocardiographic images and results are presented here. Furthermore, results from right heart catheterization and histological analyses of cardiac tissue are shown.

Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension

We describe a murine model of right ventricular pressure overload-induced by pulmonary trunk banding. Detailed protocols for intubation, surgery, and phenotyping by echocardiography are included in the paper. Custom-made instruments are used for intubation and surgery, allowing for fast and inexpensive reproduction of the model.

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary ...

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Samenvatting

Meer video's verkennen