Name: a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction
Uploaded: 2024-03-29T13:50:00.000+00:00
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Description: Watch this Scientific Journal Video about Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF at JoVE.com

We thank the Penncore and NHLBI Gene Therapy Resource Program (GTRP) for funding the generation of&#160;the Adeno-associated virus used in this project. This research was funded by grants from the National Institute of Health (NIH) (1R01HL140468) and the Miami Heart Research Institute to LS. MW was a recipient of the NIH Diversity Supplement Award from 2020 - 2022 (R01HL140468- 03S1). JH is funded by 1R01 HL13735, 1R01 HL107110, 5UM1 HL113460, 1R01 HL134558, 5R01 CA136387 (from the NIH), W81XWH-19-PRMRPCTA (from the Department of Defense), and the Starr, Lipson, and Soffer Family Foundations.

The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Miami, conforming to National Institute of Health (NIH) guidelines (IACUC protocol 23-103-ad03). For the present study, wild-type (WT) mice on 129J background were acquired from a commercial source (see Table of Materials) and bred in-house. All mice were littermates on 129J background. Experiments included both male and female mice. The LDLR/P-407 HFpEF mouse was established by administering a single dose of AAV9-cTnT-LDLR at week one and bi-weekly p407 for four weeks.
1. AAV9-cTnT-LDLR preparation and administration
NOTE: AAV9-cTNT-hLDLR plasmid (see Table of Materials) encodes the full human LDLR protein (2664bp) (Figure 1).
<ol>
	<li>AAV-LDLR viral vector preparation
	<ol>
		<li>Based on the number of animals, thaw AAV9 stock vials on ice for 20 min, then dilute AAV particles in Dulbecco&#8217;s Phosphate Buffered Saline (DPBS) to get a concentration of 1 x 1012 vector genomes/mouse in 100 &#181;L.</li>
		<li>Place the viral solution into a 28-30 G needle on a 1 mL syringe. Be careful to avoid drawing air bubbles into the needle.</li>
	</ol>
	</li>
	<li>Intravenous (i.v.) tail vein injection procedures
	<ol>
		<li>Turn on the oxygen up to 0.5 L/min and set the isoflurane anesthesia system to 4%-5%. Place the mouse in the induction chamber for ~2 min, until the animal is not responsive.</li>
		<li>Place the animal on a mouse tail illuminator restrainer (e.g., Braintree Scientific, Inc. (Braintree, MA)) and accommodate the animal lying on its side. Use isoflurane anesthesia to 2%-3% for maintenance. 
		NOTE: The heating by the restrainer device will cause dilation of the mice tail vein and, hence, make significantly easier to inject.</li>
		<li>Identify the lateral tail vein. Clean the injection area with an antiseptic using a gauze pad.</li>
		<li>Hold the end of the tail to extend it, and massage the mouse tail with fingers until the vein is visualized. Insert the needle at a low angle (10-15-degree angle) and inject 100 &#181;L of diluted AAV into the tail vein (Figure 2A).</li>
		<li>Withdraw the needle and apply pressure immediately with a finger until the bleeding stops. Return the mouse to the original cage.</li>
	</ol>
	</li>
</ol>
2. P-407 preparation and administration
<ol>
	<li>P-407 preparation
	<ol>
		<li>Prepare the solution by diluting the P-407 agent (see Table of Materials) with DPBS&#160;to a final concentration of 100 mg/mL inside a fume hood. Refrigerate the solution at 4 &#176;C overnight on a rotator to facilitate the dissolving of the P-40710.</li>
	</ol>
	</li>
	<li>Bi-weekly intraperitoneal (i.p.) injection procedure
	<ol>
		<li>Weigh each mouse on the first day of i.p. injections. Using the formula 1 g/kg, calculate the appropriate dose for each mouse according to weights and prefill syringes.</li>
		<li>Under a fume hood, manually restrain the mouse with the head and body tilted downwards in order to reposition the internal organs cranially. This technique avoids puncture of vital structures in the vicinity.</li>
		<li>Identify the left peritoneal cavity in the lower quadrant of the abdomen, lateral to the midline. Clean the site with an antiseptic.</li>
		<li>At a 45-degree angle or less, insert the needle into the peritoneal cavity (Figure 2B). Aspirate the syringe to ensure adequate insertion.</li>
		<li>If blood or tissue is present on aspiration, withdraw the needle and repeat steps 2.2.2 -2.2.4 until the syringe is clear. Dispose of the needle in the appropriate sharps container and return the mouse to the original cage.</li>
	</ol>
	</li>
</ol>
3. Echocardiography assessment
<ol>
	<li>Preparation
	<ol>
		<li>Apply depilatory cream to the mouse&#39;s chest and upper abdomen the day before or several hours before imaging. Remove the cream with wet gauze after 2 min.</li>
		<li>Anesthetize the mouse with 2.5%-3.0% isoflurane at 0.8 L/min flow rate and maintain with 1%-1.5% isoflurane. Then, secure the mouse to the appropriate platform in the supine position with the paws on electrode pads with conducting gel and cover the nose and mouth with a nose cone to ensure continuous anesthesia with isoflurane.</li>
	</ol>
	</li>
	<li>Parasternal long-axis view
	<ol>
		<li>With the mouse positioned supinely, tilt the right side of the platform 45 degrees.</li>
		<li>Next, diagonally align the transducer probe in the rail system, angling it 30-40 degrees in a clockwise manner from the right upper extremity to the left abdomen to obtain and store B-mode images.</li>
		<li>Analyze the B-mode images using ultrasound analysis software (see Table of Materials) to obtain the ejection fraction (Figure 3A).</li>
	</ol>
	</li>
	<li>Parasternal short-axis view
	<ol>
		<li>Rotate the transducer probe in the rail system 90 degrees clockwise to obtain and store B-mode and M-mode images.</li>
	</ol>
	</li>
	<li>Apical view
	<ol>
		<li>Tilt the left upper corner of the platform downwards and towards the right. Orient the transducer towards the right shoulder of the animal.</li>
		<li>Visualize the mitral valve in B-mode and color Doppler mode. Acquire and store pulsed wave (PW) Doppler and tissue Doppler images5.</li>
		<li>Analyze the PW Doppler and tissue Doppler images using the ultrasound analysis software to obtain the IVRT, E/E&#39;, and E/A (Figure 3B-E).</li>
	</ol>
	</li>
</ol>
4. Pressure-volume (PV) loop data recording
<ol>
	<li>Perform hemodynamic analyses at the conclusion of the study to assess left ventricle (LV) systolic and diastolic function, following the previously described procedure11,12.
	<ol>
		<li>Begin by inducing the mouse with isoflurane (3-5%, induction chamber).</li>
		<li>When anesthetic action starts, transfer the animal to the surgical bench, and maintain anesthesia with isoflurane (1-3%, facemask).</li>
		<li>Make a small incision in the skin over the neck to permit endotracheal intubation (through the mouth).</li>
		<li>Ventilate the animal with a mixture of oxygen and isoflurane using a rodent ventilator (e.g., Micro vent model 848, Harvard Apparatus) set at ~0.15-0.2 mL volume and the respiratory rate at 120-170 breaths/min.</li>
		<li>Monitor body temperature at ~37 &#176;C &#177; 1 &#176;C throughout the procedure using a temperature controlled surgical table.</li>
		<li>Expose and cannulate the left internal jugular vein with a 30 G needle for fluid support administration.</li>
		<li>Cut the skin over the site of the median ventral neck and expose the carotid artery. After occlusion of the distal portion of the right carotid artery, make a small cut in the artery to permit the introduction of a micro-tipped pressure-volume (PV) catheter (see Table of Materials) into the left ventricle (closed chest approach).</li>
		<li>Record PV loops during steady state and inferior vena cava occlusion.</li>
		<li>At the end of the experiment, humanely euthanize the animal (under deep anesthesia) using an approved AVMA method (e.g., Isoflurane followed by cervical dislocation).</li>
		<li>Analyze the PV data using LabChart software (see Table of Materials), and calibrate volumes using echocardiographic measurements.</li>
	</ol>
	</li>
</ol>

Investigating HFpEF Pathophysiology in a Hyperlipidemia&#45;Induced Mouse Model

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	<li>Roger, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+heart+failure:+A+contemporary+perspective.">Epidemiology of heart failure: A contemporary perspective.</a> Circ Res. 128 (10), 1421-1434 (2021).</li><li>Kosiborod, M. N., 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=Design+and+baseline+characteristics+of+step-HFpEF+program+evaluating+semaglutide+in+patients+with+obesity+hfpef+phenotype.">Design and baseline characteristics of step-HFpEF program evaluating semaglutide in patients with obesity hfpef phenotype.</a> JACC Heart Fail. 11 (8), 1000-1010 (2023).</li><li>Borlaug, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+management+of+heart+failure+with+preserved+ejection+fraction.">Evaluation and management of heart failure with preserved ejection fraction.</a> Nat Rev Cardiol. 17 (9), 559-573 (2020).</li><li>Badrov, M. B., Mak, S., Floras, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+autonomic+disturbances+in+heart+failure+with+preserved+ejection+fraction.">Cardiovascular autonomic disturbances in heart failure with preserved ejection fraction.</a> Can J Cardiol. 37 (4), 609-620 (2021).</li><li>Wu, C. 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=Myocardial+adipose+deposition+and+the+development+of+heart+failure+with+preserved+ejection+fraction.">Myocardial adipose deposition and the development of heart failure with preserved ejection fraction.</a> Eur J Heart Fail. 22 (3), 445-454 (2020).</li><li>Hahn, V. 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=Myocardial+gene+expression+signatures+in+human+heart+failure+with+preserved+ejection+fraction.">Myocardial gene expression signatures in human heart failure with preserved ejection fraction.</a> Circulation. 143 (2), 120-134 (2021).</li><li>Korolenko, T. A., 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=Early-stage+atherosclerosis+in+poloxamer+407-induced+hyperlipidemic+mice:+Pathological+features+and+changes+in+the+lipid+composition+of+serum+lipoprotein+fractions+and+subfractions.">Early-stage atherosclerosis in poloxamer 407-induced hyperlipidemic mice: Pathological features and changes in the lipid composition of serum lipoprotein fractions and subfractions.</a> Lipids Health Dis. 15, 16 (2016).</li><li>Patel, 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=Osteopontin+and+ldlr+are+upregulated+in+hearts+of+sudden+cardiac+death+victims+with+heart+failure+with+preserved+ejection+fraction+and+diabetes+mellitus.">Osteopontin and ldlr are upregulated in hearts of sudden cardiac death victims with heart failure with preserved ejection fraction and diabetes mellitus.</a> Front Cardiovasc Med. 7, 610282 (2020).</li><li>Williams, 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=Mouse+model+of+heart+failure+with+preserved+ejection+fraction+driven+by+hyperlipidemia+and+enhanced+cardiac+low-density+lipoprotein+receptor+expression.">Mouse model of heart failure with preserved ejection fraction driven by hyperlipidemia and enhanced cardiac low-density lipoprotein receptor expression.</a> J Am Heart Assoc. 11 (17), e027216 (2022).</li><li>Colly, A., Marquette, C., Courtial, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poloxamer/poly(ethylene+glycol)+self-healing+hydrogel+for+high-precision+freeform+reversible+embedding+of+suspended+hydrogel.">Poloxamer/poly(ethylene glycol) self-healing hydrogel for high-precision freeform reversible embedding of suspended hydrogel.</a> Langmuir. 37 (14), 4154-4162 (2021).</li><li>Kanashiro-Takeuchi, R. 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=Efficacy+of+a+growth+hormone-releasing+hormone+agonist+in+a+murine+model+of+cardiometabolic+heart+failure+with+preserved+ejection+fraction.">Efficacy of a growth hormone-releasing hormone agonist in a murine model of cardiometabolic heart failure with preserved ejection fraction.</a> Am J Physiol Heart Circ Physiol. 324 (6), H739-H750 (2023).</li><li>Dulce, R. A., 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=Synthetic+growth+hormone-releasing+hormone+agonist+ameliorates+the+myocardial+pathophysiology+characteristic+of+heart+failure+with+preserved+ejection+fraction.">Synthetic growth hormone-releasing hormone agonist ameliorates the myocardial pathophysiology characteristic of heart failure with preserved ejection fraction.</a> Cardiovasc Res. 118 (18), 3586-3601 (2023).</li><li>Borlaug, B. A., 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=Obesity+and+heart+failure+with+preserved+ejection+fraction:+New+insights+and+pathophysiological+targets.">Obesity and heart failure with preserved ejection fraction: New insights and pathophysiological targets.</a> Cardiovasc Res. 118 (18), 3434-3450 (2023).</li><li>Noll, N. A., Lal, H., Merryman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+heart+failure+with+preserved+or+reduced+ejection+fraction.">Mouse models of heart failure with preserved or reduced ejection fraction.</a> Am J Pathol. 190 (8), 1596-1608 (2020).</li><li>Guarnieri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considering+the+risks+and+safety+of+intraperitoneal+injections.">Considering the risks and safety of intraperitoneal injections.</a> Lab Anim (NY). 45 (4), 131 (2016).</li></ol>

JH is listed as a co-inventor on patents on GHRH analogs, which were assigned to the University of Miami and Veterans Affairs Department. JH previously owned equity in Biscayne Pharmaceuticals, the licensee of intellectual property used in this study. Biscayne Pharmaceuticals did not provide funding for this study. JH reported having a patent for cardiac cell-based therapy. He holds equity in Vestion Inc. and maintains a professional relationship with Vestion Inc. as a consultant and member of the Board of Directors and Scientific Advisory Board. JH is the Chief Scientific Officer, a compensated consultant, and an advisory board member for Longeveron and holds equity in Longeveron. JH is also the co-inventor of intellectual property licensed to Longeveron. Longeveron LLC and Vestion Inc. did not participate in funding this work. JH's relationships are disclosed to the University of Miami, and a management plan is in place.

Despite the steady increase in the prevalence of HFpEF over the last decade, a concrete understanding of the underlying pathophysiology remains elusive13. Moreover, as of today, limited evidence-based therapy exists13. An improved understanding of the mechanisms involved in cardio-metabolic HFpEF is necessary. Previously, a hyperlipidemic mouse model was introduced that mimics HFpEF with neither chronic kidney disease (CKD) nor hypertension induced by cardiac LDLR OE and p407 injections9. 
Findings revealed that the combination of cardiac LDLR OE and hyperlipidemia results in diastolic dysfunction, arrhythmias, left ventricular (LV) hypertrophy, exercise intolerance, cardiac lipid accumulation, and fibrosis in mice after four weeks, as previously published9. An increase in LDL-cholesterol uptake in the hearts, liver, and skeletal muscles and decreased triglycerides in the hearts and liver of these mice were also observed9. The advantage of this method lies in its rapidity for investigating the pathways of cardio-metabolic syndrome, which are not well understood compared to other hyperlipidemic HFpEF mouse models, such as the high-fat diet (HFD) that require up to 16 and 20 weeks to develop14. This model takes four weeks to develop and mimics metabolic abnormalities in humans. Therefore, the reproducibility of this model is essential. 
It is imperative to ensure the thorough preparation and administration of AAV9-cTnT-LDLR and P-407. The replicability of this model is highly dependent on accurate calculations of P-407 and AAV9-cTnT- LDLR concentrations and doses, as well as weight measurements. Equally important are solution preparations and proper intravenous and intraperitoneal injection techniques. Deviations in these techniques may result in significant alterations and undesired results. 
Despite the effectiveness and efficiency of this model, several limitations exist. Rigorous training is needed to perform intravenous and intraperitoneal injections. Additionally, there is a potential risk of morbidity and mortality associated with intravenous and frequent intraperitoneal injections. Mouse tail injuries may result when performing intravenous injections, while cecal puncture may occur with intraperitoneal injections, leading to peritonitis15. These injuries are typically due to incorrect techniques and may result in losing experimental subjects and treatment. Therefore, extensive training is necessary prior to performing these procedures. Another limitation is the focus of this model on the 129J strain. The rationale behind the choice of the 129J strain stems from preliminary studies that yielded more rapid diastolic dysfunction and HFpEF findings in this strain compared to the C57BL/6 mice we initially studied in unpublished investigations.
Regardless of these limitations, this model will allow for more rapid investigations into the underlying mechanisms involved in HFpEF and potential effective treatment options. Previous studies have led to the development of a pathophysiological model for cardiometabolic HFpEF-induced HFD and N[w]-nitro-l-arginine methyl ester (L-NAME) over 5-15 weeks13. However, due to the steady increase in the prevalence of HFpEF, there is an urgent need for further understanding of the pathophysiology of cardiometabolic HFpEF and the development of effective therapy. This murine model of cardiac LDLR OE and p407-induced hyperlipidemia is a rapid and feasible method of inducing cardiometabolic HFpEF for future research endeavors.

Heart failure with preserved ejection fraction (HFpEF) denotes a cardiometabolic syndrome accompanied by multiple comorbidities and constitutes over 50% of all heart failure cases1,2. Moreover, the frequency of HFpEF has steadily risen over the past decade3. With limited treatment options, HFpEF represents the most significant unmet medical necessity in cardiovascular disease, given its multifaceted pathophysiology4. Thus, an urgent need exists to enhance comprehension of the underlying mechanisms and pathophysiology of HFpEF to develop effective therapies.
Despite significant advancements in recent years, the pathophysiology of HFpEF attributed to lipotoxicity remains incompletely understood. It is established that patients with HFpEF exhibit notable myocardial lipid accumulation compared to those with heart failure with reduced ejection fraction (HFrEF) and healthy controls5. RNA sequencing data from cardiac biopsies showed downregulation of the lipoprotein lipase (LPL) gene in the HFpEF group compared to the healthy and HFrEF patients6. Poloxamer-407 (P-407) is a block co-polymer that induces hyperlipidemia by blocking LPL and subsequently increasing plasma triglycerides and low-density lipoprotein (LDL) cholesterol7. Previous studies demonstrated high LDL-Receptor (LDLR) expression in the hearts of HFpEF mice8. 
Building upon these findings and recognizing the pressing need for animal models accurately mimicking cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed and presented. This model was tailored to explore HFpEF, explicitly focusing on the involvement of lipotoxicity alongside metabolic syndrome. Induced by hyperlipidemia/LPL blockade and enhanced cardiac LDLR expression, this model was established in WT-129 mice on 129J background through biweekly intraperitoneal (i.p.) injections of P-407 combined with a single intravenous (i.v.) injection of adeno-associated virus 9-cardiac troponin T-LDLR (AAV9-cTnT-LDLR)9.
Between 4 and 8 weeks post-treatment, an extensive array of assessments was conducted, encompassing echocardiography, blood pressure recordings, whole-body plethysmography (WBP), continuous electrocardiography (ECG) telemetry, activity wheel monitoring (AWM), as well as biochemical and histological analyses9. At four weeks, the LDLR/P407 or "double treatment" mice exhibited distinct HFpEF features, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness9. Additionally, ECG telemetry and AWM revealed heart blocks and reduced activity, respectively. Notably, blood pressure and renal function remained normal9. By eight weeks, diastolic function deteriorated, and WBP measurements unveiled reduced respiratory rates9. 
Further exploration of the double treatment model revealed fibrosis, elevated wet/dry lung ratios, and heart weight/body weight ratios9. Necropsy revealed ascites, cardiac ischemia, and xanthelasmas. Intriguingly, sudden deaths were documented between 6 and 12 weeks post-treatment9. This murine hyperlipidemia-driven HFpEF model provides a rapid, valuable, and promising experimental tool for unraveling the complexities of metabolic syndrome contributing to diastolic dysfunction with lipotoxicity-mediated HFpEF.

<table><tbody><tr><td>Adeno-associated virus 9-cardiac troponin T-LDLR (AAV9-cTnT-LDLR)</td><td>U. Penn Vector Core, funded by the NHLBI Gene Therapy Program (GTRP)</td><td></td><td>Transgene plasmids and AAVs particles were generated by the U. Penn Vector Core, funded by the NHLBI Gene Therapy Program (GTRP). AAV were provided in Dulbecco&amp;rsquo;s phosphate-buffered saline (PBS) with 0.001% Pluronic F68. The Core determined AAV titers by digital droplet polymerase chain reaction (ddPCR) and assessed all preparations for capsid protein ratio by SDS-PAGE and for the presence of endotoxin. Constructs include the human (h) transcripts tagged by 3X HA, Penn Vector Core (RRID: SCR_022432). AAV9-cTNT-hLDLR plasmid encodes the full human LDLR protein (2664bp).</td></tr><tr><td>Imaging systems with a high frequency transducer probe MS400&amp;nbsp;</td><td>(VisualSonics, Toronto, ON, Canada)</td><td>Vevo 2100 or 3100</td><td></td></tr><tr><td>Isoflurane</td><td>Akorn Animal Health, Inc.</td><td>NDC: 59399-106-01</td><td></td></tr><tr><td>LabChart&amp;nbsp; software</td><td>ADInstruments</td><td>Pro version 8.1.5</td><td></td></tr><tr><td>Poloxamer 407</td><td>Sigma-Aldrich</td><td>16758</td><td></td></tr><tr><td>PV catheter</td><td>Millar Instrument</td><td>PVR 1035</td><td></td></tr><tr><td>Ultrasound analysis software&amp;nbsp;</td><td>Vevo Lab</td><td></td><td></td></tr><tr><td>Wild-type (WT) mice on 129J background&amp;nbsp;</td><td>Jackson Laboratory&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

a murine model of hyperlipidemia&#45;induced heart failure with preserved ejection fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

After 4 weeks of combined single dose i.v. AAV9-cTnT-LDLR and bi-weekly i.p. P-407 injections, echocardiography revealed HFpEF, as evidenced by the preserved ejection fraction, prolonged intraventricular relaxation time (IVRT) and E/E&#39; as well as decreased E/A (Figure 3A-E). Worse diastolic dysfunction was observed after 8 weeks when compared with data after 4 weeks. Pressure-volume (PV) loop analysis after 8 weeks of treatment showed an increased end-diastolic pressure-volume relationship slope, corroborating the echocardiography findings of diastolic dysfunction (Figure 3F). Notably, sudden death occurred in a significant number of mice treated with LDLR/P-407 between 6 and 12 weeks post-treatment with LDLR/P-407 (Figure 3G). These results indicate cardio-metabolic HFpEF, confirming the effectiveness of this protocol and experimental design. Hyperlipidemia was noted in mice treated with LDLR/P407 at 4 and 8 weeks, as evidenced by elevated total cholesterol, triglycerides, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL) cholesterol, and normal high-density lipoprotein cholesterol levels, corroborating our findings of hyperlipidemia (Figure 3H).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66442/66442fig01.jpg" /> 
Figure 1: Plasmid map for AAV9-cTnT-LDLR. <a href="https://www.jove.com/files/ftp_upload/66442/66442fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66442/66442fig02v2.jpg" /> 
Figure 2: Injection procedures. (A) Representative image demonstrating the intravenous (i.v.) tail vein injection of AAV9-cTnT-LDLR in WT mouse on 129J strain background. (B) Illustration of intraperitoneal injection of P-407 in WT mouse on 129J strain background previously treated with single i.v. dose of AAV9-cTnT-LDLR. <a href="https://www.jove.com/files/ftp_upload/66442/66442fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66442/66442fig03.jpg" /> 
Figure 3: Cardio-metabolic HFpEF. (A-E) Echocardiography parameters indicating heart failure with preserved ejection fraction (HFpEF) after 4 (n = 17) and 8 weeks (n = 11) of LDLR/P-407 treatment compared to untreated mice (n = 15). This is evidenced by preserved ejection fraction, prolonged isovolumic relaxation time (IVRT), increased&#160;E/E&#39; and reduced E/A, all indicators of diastolic dysfunction. (F) Pressure-volume loop acquisition and analyses revealed increased end-diastolic pressure-volume relationship (EDPVR) slope after 8 weeks of treatment. (G) Sudden death occurred between 6 and 12 weeks post-treatment with LDLR/P-407. (H) A lipid panel supported the findings of hyperlipidemia in mice treated with LDLR/P407 at 4 (n = 4) and 8 weeks (n = 3) as evidenced by elevated total cholesterol, triglycerides, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL) cholesterol, and normal high-density lipoprotein cholesterol levels compared to untreated mice (n = 5). The data is represented as mean &#177; SD. *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001, ****P &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/66442/66442fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF at JoVE.com

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

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Research

JoVE Journal

Medicine

1.4K Views.  University of Miami Leonard M. Miller School of Medicine. This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

Video: Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

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 ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Immunology and Infection

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics stress-induced cardiomyopathy, the chronic model, administered through an osmotic pump, mimics advanced heart failure in humans. The purpose of the described protocol is to create the chronic ISO-induced heart failure model in mice using an implanted mini-pump. This protocol has been used to induce heart failure in 100+ strains of inbred mice. Techniques on surgical pump implantation are described in detail and may be relevant to anyone interested in creating a heart failure model in mice. In addition, the weekly cardiac remodeling changes based on echocardiographic parameters for each strain and expected time to model development are presented. In summary, the method is simple and reproducible. Continuous ISO administered via the implanted mini-pump over 3 to 4 weeks is sufficient to induce cardiac remodeling. Finally, the success for ISO model creation may be assessed in vivo by serial echocardiography demonstrating hypertrophy, ventricular dilation, and dysfunction.

The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation.

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics ...

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are limited for investigating the fundamental mechanisms of HFpEF and identifying potential therapeutic targets. This work provides a detailed description of the surgical procedure of descending aortic constriction (DAC) in Tibetan minipigs to establish a large animal model of HFpEF. This model used a precisely controlled constriction of the descending aorta to induce chronic pressure overload in the left ventricle. Echocardiography was used to evaluate the morphological and functional changes in the heart. After 12 weeks of DAC stress, the ventricular septum was hypertrophic, but the thickness of the posterior wall was significantly reduced, accompanied by dilation of the left ventricle. However, the LV ejection fraction of the model hearts was maintained at &gt;50% during the 12-week period. Furthermore, the DAC model displayed cardiac damage, including fibrosis, inflammation, and cardiomyocyte hypertrophy. Heart failure marker levels were significantly elevated in the DAC group. This DAC-induced HFpEF in minipigs is a powerful tool for investigating molecular mechanisms of this disease and for preclinical testing.

The present protocol describes a step-by-step procedure to establish a minipig model of heart failure with preserved ejection fraction using descending aortic constriction. The methods for evaluating cardiac morphology, histology, and function of this disease model are also presented.

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

Biology

Murine Model for Diabetic Cardiomyopathy via High-Fat Diet and STZ Injection

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...

A Murine Model of Hyperlipidemia-Induced Heart Failure with Preserved Ejection Fraction

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