The authors acknowledge NIH K08 HL133491 for funding support.

This protocol adheres to the animal care guidelines of the University of California, Los Angeles (ARC protocol #2010-075). Readers are advised to adhere to their own IACUC-approved protocol, as peri-procedure mouse care and analgesia management may be institution-specific.
1. Preparation of isoproterenol osmotic pump
NOTE: This procedure has been applied successfully on 9+ week-old female mice weighing 18+ g from over 100 inbred mouse strains, as well as, in male mice...

<ol>
	<li>Hartupee, J., Mann, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurohormonal+activation+in+heart+failure+with+reduced+ejection+fraction.">Neurohormonal activation in heart failure with reduced ejection fraction.</a> Nature Review Cardiology. 14 (1), 30-38 (2017).</li><li>Wang, J. J., 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=Genetic+Dissection+of+Cardiac+Remodeling+in+an+Isoproterenol-Induced+Heart+Failure+Mouse+Model.">Genetic Dissection of Cardiac Remodeling in an Isoproterenol-Induced Heart Failure Mouse Model.</a> PLoS Genetics. 12 (7), 1006038 (2016).</li><li>Balakumar, P., Singh, A. P., Singh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+heart+failure.">Rodent models of heart failure.</a> Journal of Pharmacological and Toxicological Methods. 56 (1), 1-10 (2007).</li><li>Patten, R. D., Hall-Porter, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+of+heart+failure:+development+of+novel+therapies,+past+and+present.">Small animal models of heart failure: development of novel therapies, past and present.</a> Circulation: Heart Failure. 2 (2), 138-144 (2009).</li><li>Huang, W. Y., Aramburu, J., Douglas, P. S., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+expression+of+green+fluorescence+protein+can+cause+dilated+cardiomyopathy.">Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy.</a> Nature Medicine. 6 (5), 482-483 (2000).</li><li>Breckenridge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+failure+and+mouse+models.">Heart failure and mouse models.</a> Disease Model and Mechanism. 3 (3-4), 138-143 (2010).</li><li>Wittstein, I. 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=Neurohumoral+features+of+myocardial+stunning+due+to+sudden+emotional+stress.">Neurohumoral features of myocardial stunning due to sudden emotional stress.</a> New England Journal of Medicine. 352 (6), 539-548 (2005).</li><li>Templin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Features+and+Outcomes+of+Takotsubo+(Stress)+Cardiomyopathy.">Clinical Features and Outcomes of Takotsubo (Stress) Cardiomyopathy.</a> New England Journal of Medicine. 373 (10), 929-938 (2015).</li><li>Shao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+reveals+an+important+role+for+catecholamine-induced+lipotoxicity+in+the+pathogenesis+of+stress-induced+cardiomyopathy.">A mouse model reveals an important role for catecholamine-induced lipotoxicity in the pathogenesis of stress-induced cardiomyopathy.</a> European Journal of Heart Failure. 15 (1), 9-22 (2013).</li><li>Kudej, R. 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=Effects+of+chronic+beta-adrenergic+receptor+stimulation+in+mice.">Effects of chronic beta-adrenergic receptor stimulation in mice.</a> Journal of Molecular Cell Cardiology. 29 (10), 2735-2746 (1997).</li><li>Zhuo, X. Z., 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=Isoproterenol+instigates+cardiomyocyte+apoptosis+and+heart+failure+via+AMPK+inactivation-mediated+endoplasmic+reticulum+stress.">Isoproterenol instigates cardiomyocyte apoptosis and heart failure via AMPK inactivation-mediated endoplasmic reticulum stress.</a> Apoptosis. 18 (7), 800-810 (2013).</li><li>El-Demerdash, E., Awad, A. S., Taha, R. M., El-Hady, A. M., Sayed-Ahmed, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probucol+attenuates+oxidative+stress+and+energy+decline+in+isoproterenol-induced+heart+failure+in+rat.">Probucol attenuates oxidative stress and energy decline in isoproterenol-induced heart failure in rat.</a> Pharmacology Research. 51 (4), 311-318 (2005).</li><li>Rau, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+genetic+contributions+to+cardiac+pathology+induced+by+Beta-adrenergic+stimulation+in+mice.">Mapping genetic contributions to cardiac pathology induced by Beta-adrenergic stimulation in mice.</a> Circulation Cardiovascular Genetics. 8 (1), 40-49 (2015).</li><li>Berthonneche, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+response+to+beta-adrenergic+blockade+or+activation+in+23+inbred+mouse+strains.">Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains.</a> PLoS One. 4 (8), 6610 (2009).</li><li>Chang, S. C., Ren, S., Rau, C. D., Wang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoproterenol-Induced+Heart+Failure+Mouse+Model+Using+Osmotic+Pump+Implantation.">Isoproterenol-Induced Heart Failure Mouse Model Using Osmotic Pump Implantation.</a> Methods Molecular Biology. 1816, 207-220 (2018).</li><li>Shusterman, V., 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=Strain-specific+patterns+of+autonomic+nervous+system+activity+and+heart+failure+susceptibility+in+mice.">Strain-specific patterns of autonomic nervous system activity and heart failure susceptibility in mice.</a> American Journal of Physiology-Heart Circulation Physiology. 282 (6), 2076-2083 (2002).</li><li>Faulx, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+beta-adrenergic+receptor+function+influences+myocardial+responses+to+isoproterenol+stimulation+in+mice.">Strain-dependent beta-adrenergic receptor function influences myocardial responses to isoproterenol stimulation in mice.</a> American Journal of Physiology-Heart Circulation Physiology. 289 (1), 30-36 (2005).</li><li>Zhang, G. X., 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=Cardiac+oxidative+stress+in+acute+and+chronic+isoproterenol-infused+rats.">Cardiac oxidative stress in acute and chronic isoproterenol-infused rats.</a> Cardiovascular Research. 65 (1), 230-238 (2005).</li><li>Boluyt, M. O., 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=Isoproterenol+infusion+induces+alterations+in+expression+of+hypertrophy-associated+genes+in+rat+heart.">Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart.</a> American Journal of Physiology. 269 (2), 638-647 (1995).</li><li>Hohimer, A. R., Davis, L. E., Hatton, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+daily+injections+and+osmotic+pump+infusion+of+isoproterenol+cause+similar+increases+in+cardiac+mass+but+have+different+effects+on+blood+pressure.">Repeated daily injections and osmotic pump infusion of isoproterenol cause similar increases in cardiac mass but have different effects on blood pressure.</a> Canadian Journal of Physiology and Pharmacology. 83 (2), 191-197 (2005).</li><li>Cross, H. R., Murphy, E., Koch, W. J., Steenbergen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+and+female+mice+overexpressing+the+beta(2)-adrenergic+receptor+exhibit+differences+in+ischemia/reperfusion+injury:+role+of+nitric+oxide.">Male and female mice overexpressing the beta(2)-adrenergic receptor exhibit differences in ischemia/reperfusion injury: role of nitric oxide.</a> Cardiovascular Research. 53 (2), 662-671 (2002).</li><li>Klingman, G. I., McKay, G., Ward, A., Morse, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+isoproterenol+treatment+of+mice:+effects+on+catecholamines+and+rectal+temperature.">Chronic isoproterenol treatment of mice: effects on catecholamines and rectal temperature.</a> Journal of Pharmaceutical Sciences. 62 (5), 798-801 (1973).</li></ol>

The authors have nothing to disclose.

We have applied this method to over 100+ strains of inbred mice to assess cardiac outcomes due to chronic beta-adrenergic stimulation2,13. Significant differences in susceptibility to isoproterenol are known to exist among mouse strains and may be customized to the strain of interest as needed16. This may be due to the variation in beta-adrenergic receptor function between mouse strains17, strain-dependent differenc...

Heart failure with reduced ejection fraction (HFrEF) is accompanied by a well-recognized compensatory response of the sympathetic nervous system to maintain cardiovascular homeostasis1. Hemodynamic stress and deleterious effects on the heart and circulation were observed with chronic activation. These have become the cornerstone of contemporary pharmacotherapy for heart failure and are important mechanisms in the progression of heart failure and therapeutic antagonism of neurohormonal systems1.
Several mouse models are available for basic investigations of heart failure. Genetic models are attractive for exploring molecular therapies and investigating signaling pathways. However, these models may not be relevant to common forms of heart failure. Other common models include left anterior descending (LAD) artery ligation, transaortic constriction (TAC), and isoproterenol (ISO), each aiming at a different pathological etiology2,3,4,5,6. LAD artery ligation induces an anterior wall myocardial infarction thus creating a model specific for ischemic cardiomyopathy. TAC induces acute pressure overload to create a hypertensive model of heart failure. Although the pressure gradient can be measured, allowing for the stratification of hypertrophy, the acute onset of hypertension lacks direct clinical relevance4. Both LAD and TAC models require a high level of surgical expertise to execute. Acute ISO model of heart failure mimics stress-induced cardiomyopathy, also known as Takotsubo disease, which is characterized by a marked increase in catecholamines and activity in the left ventricle that mimics acute myocardial infarction7,8. In contrast, chronic ISO models of heart failure present symptoms characteristics of advanced heart failure, with chronically elevated levels of catecholamines1. The advantages of the chronic ISO model are that it provides chronic adrenergic stimulation that mimics advanced heart failure and that it is relatively easy to create. The investigator should choose a model that best recapitulates their pathology of interest.
The overall goal of this method is to induce heart failure in mice using an implanted mini pump that releases ISO continuously to mimic chronic sympathetic activation found in heart failure patients1. The method is simple and reproducible. Although there is clear variation among mouse strains, ISO administered over 3 to 4 weeks at 30 mg/kg/day is sufficient to induce cardiac remodeling in most mice. Specifically, ISO leads to a pro-hypertrophic compensatory phase during week 1 followed by wall thinning, ventricular dilation and decrease in systolic function by week 2 and 32. Success for the ISO model creation can be assessed in vivo by serial echocardiography demonstrating hypertrophy, dilation, and ventricular dysfunction, as well as ex vivo via histological and molecular assessment of harvested cardiac tissue for intramyocardial lipid accumulation, fibrosis, ER stress, apoptosis, and gene expression9,10,11,12.

<table>
	<tbody>
		<tr>
			<td>Micro-Osmotic Pump System with Flow Moderator in Place</td>
			<td>Alzet</td>
			<td>Model 1004</td>
			<td>Includes filling tube, flow moderator and pump body</td>
		</tr>
		<tr>
			<td>(-)-Isoproterenol hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>16504-1G</td>
			<td>(-)-Isoproterenol hydrochloride is a powder that needs to be stored at -20&#176;C.</td>
		</tr>
		<tr>
			<td>1 ml sterile syringe</td>
			<td>VWR</td>
			<td>BD309602</td>
			<td></td>
		</tr>
		<tr>
			<td>30 W LED Fiber optic O-ring light microscope illuminator</td>
			<td>AmScope</td>
			<td>SKU: LED-30WR</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 COATED VICRYL (polyglactin 910) Suture</td>
			<td>Ethicon</td>
			<td>J303H</td>
			<td>5-0, absorbable</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>FST</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>FST</td>
			<td>18000-46</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizers</td>
			<td>FST</td>
			<td>18000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris forceps</td>
			<td>WPI</td>
			<td>15915</td>
			<td></td>
		</tr>
		<tr>
			<td>Look Sharpoint 6-0, 18&#34; Black Nylon Monofilament Suture</td>
			<td>LOOK</td>
			<td>AA-2176</td>
			<td>6-0, non-absorbable</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>WPI</td>
			<td>15926</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline, 0.9% NaCl</td>
			<td>Fisher</td>
			<td>89167-772</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In our previously published study, we administered an ISO dosage of 30 mg/kg/d over 21 days via the osmotic pump across 105 Hybrid Mouse Diversity Panel (HMDP) strains2,13. We assessed outcomes using echocardiogram performed at baseline, week 1, 2 and 3 of ISO treatment (Figure 1). Similar to a prior study where the authors studied the impact of isoproterenol among 23 mouse strains14, we observed striking inte...

Watch this Scientific Journal Video about Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice at JoVE.com

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

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 ...

The chronic administration of isoproterenol via an osmotic pump has been widely used to recapitulate changes in advanced heart failure in mice. This method is simple and requires less surgical expertise than other models, such as LAD and TAC. Demonstrating the procedure will be Dr.Vincent Ren, a research scientist from Dr.Yibin Wang's laboratory.After weighing and recording the body weight of each mouse, calculate the appropriate amount and concentration of isoproterenol for each animal, and dissolve the isoproterenol in 120 microliters of sterile 0.9%sodium chloride. Mix thoroughly for one minute to completely solubilize the isoproterenol, and weigh and record the weight of one empty osmotic pump, together with and without its flow moderator, per mouse. Attach a 27-gauge, blunt-tipped filling tube provided with the osmotic pumps, and load one, one-milliliter syringe with 120 microliters of the isoproterenol solution per mouse.Holding a pump in the upright position, insert the filling tube through the pump opening at the top until the tip of the filling tube sits near the bottom of the pump reservoir, and slowly depress the syringe plunger until the isoproterenol solution fills to the pump opening. When the pump has been loaded, carefully remove the filling tube, and wipe off the excess solution. Insert back the flow moderator to close the pump, and wipe off any excess solution.Then reweigh the pump to confirm that over 90%of the reservoir volume has been filled. Place the mouse in the supine position on a heated pad, and remove the air from the lower abdomen. For surgical osmotic pump implantation, confirm a lack of response to toe pinch in an anesthetized mouse, and apply ointment to the animal's eyes.Clean the exposed skin with a suitable disinfectant, and make a one-centimeter midline skin incision. Use a pair of blunt-ended scissors to carefully dissect the skin from the underlying peritoneal walls, and use forceps to pull the peritoneal walls away from the underlying bowel. Use fine scissors to cut a 0.8-centimeter hole in the peritoneal wall, and insert the osmotic pump flow moderator end first into the peritoneal cavity.When the pump is in place, use 5-0 absorbable sutures in an interrupted fashion to close the hole in the peritoneal wall, and use 6-0 nonabsorbable sutures to close the skin. Then place the mouse in a dedicated incubator with monitoring until full recumbency before returning the animal to its home cage. Here, a summary of the weekly changes based on the selected echocardiographic parameters and the expected time to model development are presented.Although variations are observed between mouse strains, on average, interventricular septal wall thickness at end diastole and fractional shortening increase in the first week but decrease by later time points. The left ventricular internal diameter at end diastole and the left ventricular mass increase over a period of three weeks. Further, mouse hearts demonstrate visibly larger left ventricular chamber dimensions under isoproterenol, as well as greater amounts of fibrosis relative to saline-treated controls.It is critical to maintain a sterile surgical environment during the survival surgery to minimize post-operative infection and morbidity. Successful model creation can be assessed in vivo by serial echocardiography for hypertrophy, dilation, and dysfunction, as well as ex vivo via histological and molecular assessment of harvested cardiac tissue. We have applied this method to over 100 strains of inbred mice to assess cardiac remodeling traits due to chronic beta-adrenergic stimulation.The results are published and readily available.

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10.8K 조회수.  University of California. 삼투압 펌프를 통해 이소프로테레놀의 만성 투여는 마우스의 고급 심부전의 변화를 재구성하는 데 널리 사용되어 왔다. 이 방법은 간단하고 LAD및 TAC와 같은 다른 모델보다 적은 외과 전문 지식이 필요합니다. 이 절차를 시연하는 것은 이빈 왕의 실험실에서 연구 과학자인 빈센트 렌 박사가 될 것입니다.각 마우스의 체중을 계량 및 기록한 후 각 동물에 대해 적절한 양과...