This work was supported by Key Project Medical Science and Technology Development Foundation, Nanjing Department of Health (No.YKK16098).

Animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Nanjing Drum Tower Hospital.
1. Preparation of the reagents and animals
<ol>
	<li>Dissolve doxorubicin hydrochloride (Pfizer, USA) in sterilized water. Vortex to obtain a Dox solution of 1 mg/mL and keep at 4 &#176;C.</li>
	<li>Use C57BL/6 mice (8&#8211;10 weeks old; 25&#8211;30 g weight). For this study, mice were purchased from the Model Animal Research Center of Nanjing University and kept in the animal room of Nanjing Drum Tower Hospital.</li>
	<li>Pathogen-free mouse cages were maintained under a 12 h light/dark cycle at a constant temperature of 23 &#176;C. All animals were fed on a normal chow diet and got food and water ad libitum.</li>
</ol>
2. Establishment of DCM animal model
<ol>
	<li>Randomize mice into a normal (n = 5) group and Dox (n = 5) group.</li>
	<li>Administer the Dox solution intraperitoneally at a dose of 5 mg/kg using a 1 mL sterilized syringe 1x a week for the Dox group. Treat the control mice in the same way with the same amount of saline solution.</li>
	<li>Measure the body weight of the two groups weekly. Accordingly, adjust the injection dose based on body weight weekly for a total of 4 weeks, with a cumulative dose of 20 mg/kg (Figure 1). 
	NOTE: A time period of 4 weeks was chosen because echocardiography at 4 weeks showed a significant difference in cardiac function between the two groups.</li>
</ol>
3. Echocardiographic examination
<ol>
	<li>At the end of the fourth week, conduct an echocardiographic examination of the mice.</li>
	<li>Anesthetize the mice with 2% isoflurane intranasally. If the mice do not respond to a pinch of the skin with a toothed tweezer or stimulating the toes and tails, continue with the protocol.</li>
	<li>Place the mouse on an animal-handling platform in the supine position. To maintain the anesthesia, cover the animal&#39;s nose and mouth with a nose cone and deliver 2% isoflurane. 
	NOTE: For IP anesthesia, inject 4% chloral hydrate at a dose of 0.2 mL/20 g.</li>
	<li>Remove chest fur carefully with an electric shaver. Assess cardiac function in vivo using transthoracic echocardiography.</li>
	<li>Perform the LV echocardiogram in both the parasternal long-axis and short-axis views at a frame rate of 233 Hz. End-systolic and end-diastolic dimensions were defined as the phases corresponding to the ECG T wave and to the R wave, respectively.</li>
	<li>On the M-mode tracings, measure the average LV end-systolic diameter (LVIDd), LV end-diastolic diameter (LVIDs), interventricular septal thickness (IVS), and LV posterior wall thickness (LVPW) from 3&#8211;5 heart beats. Also calculate the ejection fraction (EF) and fraction shortening (FS) based on echocardiography.</li>
</ol>
4. Histological staining
<ol>
	<li>After the echocardiographic analysis, sacrifice the mice by intraventricular injection of 10% potassium chloride.</li>
	<li>Perfuse the heart with about 30 mL of saline after dissection until the liver and lung become pale.</li>
	<li>Excise the heart and thoroughly wash it in phosphate buffer solution to extrude blood.</li>
	<li>Fix the heart in 4% formalin at room temperature for 24 h and treat the tissue in a paraffin box so that the paraffin wax cools down and solidifies.</li>
	<li>Cut the hearts into 5 &#956;m thick slices for pathological staining.</li>
	<li>Dewax and rehydrate sections containing papillary muscle.
	<ol>
		<li>Incubate slides at 55 &#176;C for 30 min. Then, incubate in xylene 2x for 2 min each; 100% ethanol 2x for 2 min each; 95% ethanol 2x for 2 min each; 80% ethanol for 2 min; 75% ethanol for 2 min; and 50% ethanol for 2 min.</li>
	</ol>
	</li>
	<li>Stain using hematoxylin and eosin (H&#38;E) as well as Masson&#8217;s stain.</li>
</ol>

<ol>
	<li>Weintraub, R. G., Semsarian, C., MacDonald, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilated+cardiomyopathy.">Dilated cardiomyopathy.</a> Lancet. 390 (10092), 400-414 (2017).</li><li>Ichihara, 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=Attenuation+of+oxidative+stress+and+cardiac+dysfunction+by+bisoprolol+in+an+animal+model+of+dilated+cardiomyopathy.">Attenuation of oxidative stress and cardiac dysfunction by bisoprolol in an animal model of dilated cardiomyopathy.</a> Biochemical and Biophysical Research Communications. 350 (1), 105-113 (2006).</li><li>Fountoulakis, 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=Alterations+in+the+heart+mitochondrial+proteome+in+a+desmin+null+heart+failure+model.">Alterations in the heart mitochondrial proteome in a desmin null heart failure model.</a> Journal of Molecular and Cellular Cardiology. 38 (3), 461-474 (2005).</li><li>Fairweather, D., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coxsackievirus-induced+myocarditis+in+mice:+a+model+of+autoimmune+disease+for+studying+immunotoxicity.">Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity.</a> Methods. 41 (1), 118-122 (2007).</li><li>Wang, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+therapeutic+anti-CD4+monoclonal+antibody+inhibits+T+cell+receptor+signal+transduction+in+mouse+autoimmune+cardiomyopathy.">A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy.</a> Chinese Medical Journal. 120 (15), 1319-1325 (2007).</li><li>Riad, 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=Toll-like+receptor-4+deficiency+attenuates+doxorubicin-induced+cardiomyopathy+in+mice.">Toll-like receptor-4 deficiency attenuates doxorubicin-induced cardiomyopathy in mice.</a> European Journal of Heart Failure. 10 (3), 233-243 (2008).</li><li>Kankeu, C., Clarke, K., Passante, E., Huber, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+chronic+dilated+cardiomyopathy-the+apoptosis+hypothesis+revisited.">Doxorubicin-induced chronic dilated cardiomyopathy-the apoptosis hypothesis revisited.</a> Journal of Molecular Medicine. 95 (3), 239-248 (2017).</li><li>Zhao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-140-5p+aggravates+doxorubicin-induced+cardiotoxicity+by+promoting+myocardial+oxidative+stress+via+targeting+Nrf2+and+Sirt2.">MicroRNA-140-5p aggravates doxorubicin-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2.</a> Redox Biology. 15, 284-296 (2018).</li><li>O'Connell, J. 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No conflicts of interest declared.

Dox is a nonspecific periodic antitumor chemotherapy drug commonly used in clinical practice12. Its main side effect is cardiotoxicity, characterized by cardiomyopathy and subsequent heart failure13. The underlying mechanism includes damage of myocardial lipid peroxidation, inhibition of myocardial sarcoplasmic reticulum Ca2+-ATPase activity, and activation of the myocardial local renin-angiotensin system, resulting in increased AT II production and intracellular calcium overload14. Due to the lethal effect of a large dose of intraperitoneal injection of Dox, an alternative way to establish a reliable model was used in this protocol.
The DCM model established by this method is very similar to the pathogenesis of human DCM. In addition to heart failure, the most common complication of Dox, ascites were observed in DCM mice. Therefore, during model development, attention should be paid to sterile operation and timely replacement of syringes. In addition, for the Dox dose, two methods of administration were compared: a long course of treatment using a small dose, and short-term treatment with a high dose. The results show that continuous injection of Dox at 5 mg/kg for four weeks can result in an accurate model of DCM, lower mice mortality, and a shorter time for model establishment.
In summary, a stable, reliable, and economical mouse DCM animal model can be successfully established by intraperitoneal injection of Dox. This research provides an ideal model for studying DCM. It will contribute to reveal the pathogenesis of dilated cardiomyopathy or chronic heart failure and explore new drugs or treatments for future clinical applications.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.
Figure 1 was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v2.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61158/61158fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v5.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61158/61158fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.

Erratum: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

One of the most common causes of heart failure, DCM is characterized by ventricular dilatation and decreased cardiac function and is the most common reason for heart transplantation worldwide1. In order to further investigate its pathogenesis and find effective treatments, access to mature animal models is especially important. The purpose of the described experiments is to establish a stable mouse model of DCM that resembles human DCM.
Due to the complex pathogenesis of DCM, there are many different methods to make corresponding animal models. Spontaneous DCM models2 are relatively stable, but they are expensive and not easily available. Genetically modified animal models3 are not well-established and require more experimental use. DCM animal models induced by viral infection4 or autoimmune defects5 are easy to obtain, but they are not wholly representative of DCM. Models associated with myocardial toxicity include alcohol-induced DCM models and Dox-induced DCM animal models.
The Dox-induced cardiomyopathy model is obtained by intraperitoneal injection of Dox6. The model exploits the most severe chronic side effect of Dox: After Dox exposure, patients develop late onset DCM symptoms with clinical uniformity7. Dox-induced oxidative stress8 and mitochondrial damage9, which lead to cardiomyocyte apoptosis, are symptoms in the pathogenesis of DCM. There are acute and chronic Dox treatment models: a single high dose of Dox (15 mg/kg) induces a short-term model for cardiomyopathy10, while repetitive low-dose Dox injections (six&#160;weekly, 3&#160;mg/kg) induce a long-term model for cardiomyopathy11. Based on the presented study, wild type mice intraperitoneally injected once a week for a month at a dose of 5 mg/kg display morphology and histology of the heart consistent with the characteristics of DCM by the end of the treatment, providing an ideal way to establish a DCM model.

<table><tbody><tr><td>4% paraformaldehyde</td><td>servicebio</td><td>CAS30525-89-4</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Model Animal Research Center of Nanjing University</td><td>\</td><td></td></tr><tr><td>Doxorubicin hydrochloride</td><td>Pfizer</td><td>CAS25316-40-9</td><td></td></tr><tr><td>echocardiography</td><td>Visualsonics</td><td>\</td><td></td></tr><tr><td>Hematoxylin and Eosin staining kit</td><td>Solarbio</td><td>G1120</td><td></td></tr><tr><td>Masson staining kit</td><td>Solarbio</td><td>G1343</td><td></td></tr><tr><td>phosphate buffer solution</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>potassium chloride</td><td>Sigma</td><td>CAS7447-40-7</td><td></td></tr><tr><td>sterilized syringe</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr></tbody></table>

a doxorubicin-induced murine model of dilated cardiomyopathy in vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Cardiac function 
Dilated cardiomyopathy is characterized by progressive ventricular dilatation and contractile dysfunction. Figure 2 shows representative echocardiographic images of the two groups. Dox-treated mice showed markedly reduced left ventricular ejection fraction and left ventricular fractional shortening (Figure 3A,B). The LV diameter also increased in both the diastolic and systolic phases (Figure 3C,D). This revealed that Dox-treated mice had an impaired cardiac function.
Histological staining 
Pathological staining was performed to observe the pathological changes of saline-treated mice and Dox-treated mice. Myocardial fibers of the mice in the control group were neatly arranged, without infiltrating leukocytes. In the Dox group, myocardial myofibers were disordered and broken, and cardiomyocytes were smaller and thinner (Figure 4A). Masson staining showed that Dox-treated mice had more interstitial fibrosis (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v5.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61158/61158fig1v5large.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/61158/61158fig2v2.jpg" /> 
Figure 2: The echocardiography of the two groups.&#160;(A) Control group; (B) Dox group. Left is the long-axis section and right is the short-axis section. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61158/61158fig2v2large.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/61158/61158fig3v2.jpg" /> 
Figure 3: The cardiac function variables between groups.&#160;(A) EF; (B) FS; (C) LVEDd; (D) LVEDs. *P &#60; 0.05, Student&#8217;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61158/61158fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61158/61158fig4v2.jpg" /> 
Figure 4: Histological staining.&#160;(A) H&#38;E staining; (B) Masson staining. Left is the control group and right is the Dox group. The upper arrow shows that Dox-treated cardiomyocytes were smaller and thinner and the myofibers were disordered and broken. The lower arrow denotes the presence of interstitial fibrosis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61158/61158fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5

Watch this Scientific Journal Video about A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo at JoVE.com

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

a-doxorubicin-induced-murine-model-of-dilated-cardiomyopathy-in-vivo

Nanyang Technological University

Research

JoVE Journal

Medicine

4.4K Views.  Clinical College of Nanjing Medical University. Beschreven is een protocol om een doxorubicine-geïnduceerd gedilateerde cardiomyopathie (DCM) model vast te stellen bij muizen via langdurige intraperitoneale injectie van Doxorubicine.

Video: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Developmental Biology

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 ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Bioengineering

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

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 ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

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 Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

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