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

<ol>
	<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8217;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&#160;</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&#160; 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&#160;</td>
			<td>Vevo Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wild-type (WT) mice on 129J background&#160;</td>
			<td>Jackson Laboratory&#160;</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 ...

a-murine-model-hyperlipidemia-induced-heart-failure-with-preserved

Research

JoVE Journal

Medicine

1.3K 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 

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...