This work was supported by the Natural Science Funds of Zhejiang Province (LQ22H020010).

All the experiments were conducted in accordance with the guidelines for the care and use of laboratory animals at Zhejiang University and were approved by the Animal Advisory Committee at Zhejiang University.
1. Left anterior descending coronary artery ligation (LAD ligation) surgery
NOTE: Eight week old male C57BL/6J mice were used as models. The hearts were harvested 2 weeks after MI. Left anterior descending coronary artery ligation surgery was carried out as previously described and demonstrated11.
<ol>
	<li>Disinfect the surgical instruments with 70% ethanol before surgery.</li>
	<li>Weigh the mouse, and then anesthetize the mouse with intraperitoneal injections of 1% pentobarbital sodium (50 mg/kg). Observe the toe-pinch reflex, and measure the respiratory rate to evaluate the depth of anesthesia during the procedure.</li>
	<li>Place the mouse on the operating table with a 37 &#176;C heating pad. Use ophthalmic ointment to prevent eye dehydration.</li>
	<li>Depilate its left precordial chest and neck with depilatory cream, and use cotton swabs to remove hairs. Disinfect its skin with iodophor, followed by 70% alcohol to clean the area three times.</li>
	<li>Perform a midline cervical incision (0.8-1.0 cm) under a microscope. Separate the skin, muscle, and tissue surrounding the trachea, and then insert a 20 G cannula into the trachea. 
	NOTE: Observe while inserting the cannula into the trachea under microscopic view to ensure that the tube is not inserted into the esophagus.</li>
	<li>Connect the mouse to a rodent ventilator set at 0.2 mL tidal volume with 150 strokes per minute.</li>
	<li>Incise the mouse chest obliquely along the midclavicular line with a sterile scissor. Dissect the subcutaneous tissue to expose the ribs.</li>
	<li>Use sterile scissors to cut the skin obliquely along the left mid-clavicular line (0.8-1.0 cm). Perform left thoracotomy to separate the third and fourth ribs to expose the heart. Use a rib retractor for a clear view.</li>
	<li>Open the pericardium with curved forceps, and then ligate the left anterior descending artery (below the left atrium appendage) using a 7-0 silk suture. A pale anterior wall of the left ventricle indicates that the myocardial perfusion is successfully interrupted.</li>
	<li>Release the rib retractor, and stitch the thoracic incision in layers with a 5-0 silk suture while expelling air from the chest. Stitch the neck incision with a 5-0 suture silk suture.</li>
	<li>Remove the tracheal tube from the mouse once its spontaneous breathing is restored. Intraperitoneally inject 0.5 mL of pre-warmed sterile saline. Inject buprenorphine (0.1 mg/kg) every 12 h subcutaneously to minimize pain.</li>
	<li>Finally, place the mouse on the heating pad to wait for resuscitation. Monitor the mouse to ensure it does not suffer from excessive dyspnea or hemorrhaging via visual observation.</li>
	<li>Return the mouse to its cage after it wakes up. For the first week, monitor the mouse daily to verify that its mobility, grooming, and eating habits are adequate. According to the experiment&#39;s needs, harvest the heart at different time points after MI. 
	&#8203;NOTE: In this protocol, the single-cell suspension was prepared with the heart 2 weeks after MI.</li>
</ol>
2. Preparation of the cardiac single-cell suspension
<ol>
	<li>Preparation of the solutions and media
	<ol>
		<li>Cardiac tissue dissociation buffer: Mix 10 mg of collagenase IV (600 U/mL), 5 mg of dispase II (0.5 U/mL), and 50 &#181;L of DNase I (100 &#181;g/mL) in 5 mL of RPMI 1640. Pre-warm this medium to 37 &#176;C in a water bath before use.</li>
		<li>Cell wash buffer: Prepare 1% BSA or 10% FBS in phosphate-buffered saline (PBS, PH 7.4), and keep at 4 &#176;C or on ice.</li>
		<li>Perfusion solution: Pre-cool PBS (PH 7.4) as a perfusion solution.</li>
		<li>Red blood cell (RBC) lysis buffer: Use the RBC lysis buffer as mentioned by the manufacturer. 
		NOTE: Refer to the Table of Materials for the list of reagents and equipment used in this study.</li>
	</ol>
	</li>
	<li>Euthanize the mouse by an intraperitoneal pentobarbital sodium (150 mg/kg) injection.</li>
	<li>Heart dissection
	<ol>
		<li>Place the mouse in a supine position by fixing its hindlimbs in place with adhesive tape.</li>
		<li>Spray the body with 70% ethanol, and then cut vertically through the abdominal skin and muscles while avoiding piercing the liver. Carefully open the thorax while avoiding puncturing the heart.</li>
		<li>Use the pre-cooled PBS (PH 7.4) to perfuse the left ventricle until the colorless perfusion fluid is observed (approximately 15 mL of PBS [pH 7.4] is needed).</li>
		<li>Gently lift the heart from the thorax with forceps, and cut away the excess tissue attached to the outside of the heart with ophthalmic scissors. Place the heart in 1 mL of pre-cooled PBS (PH 7.4) in a 1.5 mL microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Enzymatic dissociation of the heart tissue
	<ol>
		<li>Transfer the mouse heart into one well of a 24-well plate with 150 &#181;L of the cardiac tissue dissociation buffer placed on ice, and cut it into small pieces (~1 mm) with a scissor.</li>
		<li>Transfer the minced heart tissue into a 15 mL centrifuge tube with 5 mL of the tissue dissociation buffer. 
		NOTE: The recommended cardiac tissue dissociation buffer volume is 5 mL/heart.</li>
		<li>Place the centrifuge tube in the electrically thermostatic reciprocating shaker at 125 rpm for 1 h at 37 &#176;C. Pipet the tissue suspension up and down 10 times every 20 min.</li>
		<li>Filter the cell suspension through a 70 &#181;m cell strainer to remove undigested tissue and clumps. Add 25 mL of the cell wash buffer to rinse the original tube, and then transfer it to rinse the cell strainer. Next, filter the cell suspension through a 40 &#181;m cell strainer to further remove cell clumps.</li>
		<li>Centrifuge the filtered cell suspension at 300 x g for 5 min at 4 &#176;C. Discard the supernatant, and then resuspend the cell sample in 1x RBC lysis buffer for 5 min at room temperature (RT).</li>
		<li>Add four times more volume of the cell wash buffer, and centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant, and resuspend the cells in 10 mL of the cell wash buffer. Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant, and resuspend the cells in 1 mL of the cell wash buffer.</li>
		<li>Mix 18 &#181;L of the cell suspension with 2 &#181;L of acridine orange (AO)/propidium iodide (PI) staining solution (9:1), and add 10 &#181;L of the mixture to a counting chamber slide. Evaluate the cell numbers and viability using an automatic cell counter.</li>
	</ol>
	</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+revisited:+Inflammation+versus+resolution+of+inflammation+following+myocardial+infarction.">Inflammation revisited: Inflammation versus resolution of inflammation following myocardial infarction.</a> Basic Research in Cardiology. 109 (6), 444 (2014).</li><li>Tzahor, E., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+coalition+to+heal-the+impact+of+the+cardiac+microenvironment.">A coalition to heal-the impact of the cardiac microenvironment.</a> Science. 377 (6610), 4443 (2022).</li><li>Song, 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=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485 (7400), 599-604 (2012).</li><li>Jaitin, D. 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The authors declare that they have no competing interests.

This article aimed to describe a protocol to isolate single non-cardiomyocytes from mouse hearts post MI. The protocol can be applied to isolate different types of cells in the post-MI microenvironment with high quality, including immune cells, endothelial cells, and fibroblasts. Three essential factors are crucial for obtaining a high-quality cell suspension for single-cell sequencing. The first one is the setting of the enzymatic digestion. It is important to control the time of digestion and the volume and concentration of the digestive enzymes to assure both cell viability and isolation efficiency. In this case, we appropriately increased the concentration of the enzyme and prolonged the digestion time. Here, we recommend a digestion duration of 45 min to 1 h. A shorter duration may increase the cell viability but may lead to insufficient cardiac tissue digestion and may increase the amount of cell clumps. In addition, we used DNase I to prevent cell aggregation because lytic cells can release free DNA to wrap the surrounding cells to form cell clumps13.&#160;The second essential factor is using a cell washing solution containing BSA or FBS. BSA or FBS can not only protect the activity of the enzymes but also improve the viability of the cells. The final essential factor is facilitating FACS to eliminate cell fragments and dead cells. Cell fragments can result in an inaccurate cell count and viability. These factors together enhance the viability and purity of the cell suspension, making it more compatible with single-cell sequencing.
This protocol firstly describes the cell suspension preparation for single-cell sequencing, and it also includes additional procedures to enhance the quality of the cell suspension at a relatively low cost. In comparison with other protocols used by previous studies, we avoid using digestive enzymes that can cause the aggregation of cells induced by free DNA, such as trypsin14. The removal of the cell debris is one of the major challenges for single-cell studies, and some studies use dead cell removal kits to improve cell viability before running the samples14,15. However, here, we suggest using FACS to enhance the sample purity due to its high effectiveness in reducing dead cells and debris.
The single-cell suspension obtained by this protocol can be further analyzed by flow analysis or primary cell culture (standard aseptic procedures)18. As the digestive enzymes are relatively mild, it is suitable to digest cardiac tissue taken in different stages of MI, as well as that from healthy hearts. However, this protocol has certain limitations. For example, prolonged digestion at 37 &#176;C inevitably promotes the expression of stress genes19,20. This limitation can be solved by using low-temperature active protease or reducing the digestion time followed by FACS. Also, this protocol does not include isolating cardiomyocytes, and, thus, it is not suitable for studies on local tissue responses.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, and its mortality increasing throughout the world1. MI is caused by an insufficient blood supply to the surrounding myocardium, which can be a result of a coronary artery blockage that occurs with atherosclerotic plaque rupture. Although percutaneous coronary intervention (PCI) has reduced the mortality rates of acute MI patients, the high prevalence of heart failure post MI remains a problem2. The key pathophysiology underlying post-MI heart failure is the body&#39;s compensatory response to cardiac injuries, which involves replacing the dead cardiac muscle with non-contractile fibrotic scars. These adaptive responses significantly rely on local inflammation mediated by the interactions between multiple cell types and cardiac tissue cells, and this inflammation is now considered as a potential therapeutic target to reduce fibrotic scar formation and, thus, protect against post-MI heart failure3,4. Interestingly, the microenvironment at the infarction site experiences a time-dependent transition in the infiltrating cell types and functions at different stages of MI1,5. Many studies have shown that non-cardiomyocytes (e.g., immune cells, fibroblasts, and endothelial cells) play central roles in post-MI inflammation and tissue repair5,6. In recent years, single-cell sequencing has been widely used as a powerful tool for elucidating the involvements and functions of non-cardiomyocytes in the post-MI microenviroment7,8. This provides insights into the pathophysiology of post-MI injury and repair and the development of potential therapies against post-MI heart failure.
High-throughput RNA sequencing (RNA-Seq) is a technique used to study entire transcriptomes in great detail using next-generation sequencing (NGS)7,8,9. Recently, the development of scRNA-Seq has revolutionized the biomedical research field. Compared with conventional bulk sequencing, scRNA-Seq analyses gene expression profiles and transcriptional heterogeneity at the single-cell level7,8. This technique significantly promotes the research of the cellular pathophysiology of MI9,10 by identifying different circulating cell types in the post-MI microenvironment and uncovering the interaction between cardiomyocytes and non-cardiomyocytes. These findings further contribute to uncovering novel therapeutic targets for post-MI heart failure. In general, the scRNA-seq-based post-MI experiment includes three main sections: (1) the establishment of post-MI animal model; (2) the preparation of the cell suspension; and (3) sample sequencing and data analysis. Noticeably, preparing the cell suspension is the most critical step in the preparation of the scRNA-Seq experiment because the quality of the cell suspension determines the accuracy of the results.
This protocol is designed to extract a non-cardiomyocyte cell suspension from the post-MI cardiac tissue; significantly, the specific details for maintaining cell viability and resolution are included. Meanwhile, the equipment used in this protocol, such as surgical kits for mice, rodent ventilators, and centrifuges, can be found in most animal experiment centers and biomedical laboratories, and, thus, the experiment cost of this protocol is relatively low. Furthermore, if considering time points and sites of infarction as variables, this protocol can be applied to simulate a wide range of clinical scenarios, especially for the post-MI complications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acridine Orange / Propidium Iodide Stain</td>
			<td>Logos biosystems</td>
			<td>F23001</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated&#160;Cell&#160;Counter</td>
			<td>Logos biosystems</td>
			<td>L20001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Servicebio</td>
			<td>G5001</td>
			<td>Cytoprotective effect</td>
		</tr>
		<tr>
			<td>Cell Counting Slides</td>
			<td>Logos biosystems</td>
			<td>L12001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td>Digestive enzymes</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma</td>
			<td>D4693</td>
			<td>Digestive enzymes</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td>Prevent cell clumping</td>
		</tr>
		<tr>
			<td>Falcon&#160;40&#956;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td>Remove cell clumps</td>
		</tr>
		<tr>
			<td>Falcon&#160;70&#956;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td>Remove undigested tissue and clumps</td>
		</tr>
		<tr>
			<td>Flow Cell Sorter</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodophor</td>
			<td>OU QING SI</td>
			<td>10054963976859</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>FST</td>
			<td>12061-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic Forceps</td>
			<td>RWD</td>
			<td>F14012-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic Scissors</td>
			<td>RWD</td>
			<td>S11036-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline&#160;</td>
			<td>Servicebio</td>
			<td>G4202-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer</td>
			<td>Beyotime</td>
			<td>C3702-120ml</td>
			<td>Remove red blood cells</td>
		</tr>
		<tr>
			<td>Rib Retractor</td>
			<td>FST</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Ventilator</td>
			<td>Harvard</td>
			<td>730043</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>Solvent&#160;solution of enzyme</td>
		</tr>
		<tr>
			<td>Sterile Scissor</td>
			<td>RWD</td>
			<td>S14014-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of a non-cardiomyocyte cell suspension for single-cell rna sequencing from a post-myocardial infarction adult mouse heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

A single-cell suspension with high vitality was obtained by implementing section 2 of this protocol. However, cell fragments could still be observed (Figure 1A); hence, fluorescence-activated cell sorting (FACS) was performed to further improve the quality16.After FACS, the average cell size reduces from 9.6 &#181;m to 9.1 &#181;m (Table 1), which suggests that the proportion of cell fragments can be effectively reduced in the cell suspension by FACS (Figure 1B). The gating strategy used for FACS is shown in Figure 1C.
In addition to the images in Figure 1, the cell concentration, average cell size, and cell viability were measured by a cell counter (Table 1). The differences in cell concentrations were due to the volume of the cell suspension. Noticeably, the average cell size decreased after FACS (Table 1), which shows that FACS can effectively reduce cell clumping and remove cell fragments.
ScRNA-seq was performed to study the cardiac immunological microenvironment while verifying the cell suspension quality obtained through this protocol. After preparing the single-cell suspension, we firstly sorted the cells that expressed surface protein CD45, which is a common marker for a wide range of immune cells. Next, we performed scRNA-seq on CD45+ cells using the 10X Genomics platform17. In this way, we pooled 14,217 individual cells from three healthy heart samples into a merged data set after quality control (Table 2). The unsupervised clustering and reduction in t-distributed stochastic neighbor embedding (t-SNE) dimensionality showed an obvious separation of seven types of immune cells (Figure 2). Moreover, each immune cell type showed a high expression of classic markers. Conclusively, these results show that the single-cell suspension prepared by this protocol has a high potency for single-cell sequencing.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64290/64290fig01.jpg" /> 
Figure 1: Cell viability in the cardiac single-cell suspension. (A)&#160;Cell viability without FACS. The black arrowheads indicate cell fragments (no fluorescence). (B) Cell viability with FACS. The yellow arrow indicates live cells (green fluorescence), and the red arrow indicates dead cells (red fluorescence).(C) Gating strategy. Scale bar: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64290/64290fig01large.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/64290/64290fig02v3.jpg" /> 
Figure 2: Single-cell RNA sequencing of cardiac CD45+ cells from three mouse heart samples. (A)&#160;tSNE plot of CD45+ cells showing the immune cell populations identified based on canonical marker genes. (B) Heatmap of the top five differentially expressed genes in each subpopulation. <a href="https://www.jove.com/files/ftp_upload/64290/64290fig02largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Before FACS</td>
			<td>After FACS</td>
		</tr>
		<tr>
			<td>Total cell concentration (cells/mL)</td>
			<td>1.51 x 106&#160;</td>
			<td>3.40 x 105</td>
		</tr>
		<tr>
			<td>Live cell concentration (cells/mL)</td>
			<td>1.40 x 106&#160;</td>
			<td>3.15 x 105</td>
		</tr>
		<tr>
			<td>Dead cell concentration (cells/mL)</td>
			<td>1.09 x 105</td>
			<td>2.48 x 104</td>
		</tr>
		<tr>
			<td>Viability (%)</td>
			<td>92.8</td>
			<td>92.7</td>
		</tr>
		<tr>
			<td>Average cell size (&#956;m)</td>
			<td>9.6</td>
			<td>9.1</td>
		</tr>
	</tbody>
</table>
Table 1: Cardiac single-cell suspension before and after FACS. The cell densities of the cardiac single-cell suspension obtained before and after FACS are compared using an automated cell counter.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Estimates</td>
		</tr>
		<tr>
			<td>Estimated Number of Cells</td>
			<td>14,217</td>
		</tr>
		<tr>
			<td>Mean Reads per Cell</td>
			<td>81,017</td>
		</tr>
		<tr>
			<td>Median Genes per Cell</td>
			<td>1,374</td>
		</tr>
		<tr>
			<td>Total Genes Detected</td>
			<td>18,424</td>
		</tr>
		<tr>
			<td>Median UMI Counts per Cell</td>
			<td>4,021</td>
		</tr>
		<tr>
			<td>Valid Barcodes</td>
			<td>98.4%</td>
		</tr>
		<tr>
			<td>Reads Mapped to Genome</td>
			<td>95.1%</td>
		</tr>
	</tbody>
</table>
Table 2: Quality control statistics. The table lists the different estimates of the single-cell data of healthy mouse hearts.

Watch this Scientific Journal Video about Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart at JoVE.com

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

preparation-non-cardiomyocyte-cell-suspension-for-single-cell-rna

Universidad San Sebastian

Research

JoVE Journal

Medicine

1.9K Views.  Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province. Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Video: Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

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 ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

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 ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development.
Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult G&#246;ttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model.
MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in G&#246;ttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in G&#246;ttingen minipigs and at 2 months in Landrace pigs, respectively.
Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in G&#246;ttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, G&#246;ttingen minipigs showed a slight increase in LVED mass only at 6 months.
In summary, this is the first characterization of post-MI HF in G&#246;ttingen minipigs in comparison to Landrace pigs, showing that the G&#246;ttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

The overall goal of the current study is to present the techniques of induction of myocardial infarction (MI) and post-myocardial infarction heart failure (post-MI HF) in closed-chest, adult G&#246;ttingen minipigs and the characterization of post-MI HF model in G&#246;ttingen minipigs as compared to Landrace pigs.

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...