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 ca...

<ol>
	<li>Reed, G. W., Rossi, J. E., Cannon, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction.">Acute myocardial infarction.</a> Lancet. 389 (10065), 197-210 (2017).</li><li>Gu, 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=Incident+heart+failure+in+patients+with+coronary+artery+disease+undergoing+percutaneous+coronary+intervention.">Incident heart failure in patients with coronary artery disease undergoing percutaneous coronary intervention.</a> Frontiers in Cardiovascular Medicine. 8, 727727 (2021).</li><li>Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+response+in+myocardial+injury,+repair,+and+remodelling.">The inflammatory response in myocardial injury, repair, and remodelling.</a> Nature Reviews. Cardiology. 11 (5), 255-265 (2014).</li><li>Kain, V., Prabhu, S. D., Halade, G. 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. 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=Massively+parallel+single-cell+RNA-seq+for+marker-free+decomposition+of+tissues+into+cell+types.">Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.</a> Science. 343 (6172), 776-779 (2014).</li><li>Massaia, 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=Single+cell+gene+expression+to+understand+the+dynamic+architecture+of+the+heart.">Single cell gene expression to understand the dynamic architecture of the heart.</a> Frontiers in Cardiovascular Medicine. 5, 167 (2018).</li><li>Papalexi, E., Satija, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+to+explore+immune+cell+heterogeneity.">Single-cell RNA sequencing to explore immune cell heterogeneity.</a> Nature Reviews. Immunology. 18 (1), 35-45 (2018).</li><li>Jin, 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=Single-cell+RNA+sequencing+reveals+the+temporal+diversity+and+dynamics+of+cardiac+immunity+after+myocardial+infarction.">Single-cell RNA sequencing reveals the temporal diversity and dynamics of cardiac immunity after myocardial infarction.</a> Small Methods. 6 (3), 2100752 (2022).</li><li>Tombor, L. 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=Single+cell+sequencing+reveals+endothelial+plasticity+with+transient+mesenchymal+activation+after+myocardial+infarction.">Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction.</a> Nature Communications. 12 (1), 681 (2021).</li><li>Virag, J. A., Lust, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+artery+ligation+and+intramyocardial+injection+in+a+murine+model+of+infarction.">Coronary artery ligation and intramyocardial injection in a murine model of infarction.</a> Journal of Visualized Experiments. (52), e2581 (2011).</li><li>Reichard, A., Asosingh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practices+for+preparing+a+single+cell+suspension+from+solid+tissues+for+flow+cytometry.">Best practices for preparing a single cell suspension from solid tissues for flow cytometry.</a> Cytometry Part A. 95 (2), 219-226 (2018).</li><li>Gladka, M. 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=Single-cell+sequencing+of+the+healthy+and+diseased+heart+reveals+cytoskeleton-associated+protein+4+as+a+new+modulator+of+fibroblasts+activation.">Single-cell sequencing of the healthy and diseased heart reveals cytoskeleton-associated protein 4 as a new modulator of fibroblasts activation.</a> Circulation. 138 (2), 166-180 (2018).</li><li>Garcia-Flores, 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=Preparation+of+single-cell+suspensions+from+the+human+placenta.">Preparation of single-cell suspensions from the human placenta.</a> Nature Protocols. , (2022).</li><li>Guez-Barber, 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=FACS+purification+of+immunolabeled+cell+types+from+adult+rat+brain.">FACS purification of immunolabeled cell types from adult rat brain.</a> Journal of Neuroscience Methods. 203 (1), 10-18 (2012).</li><li>Rheinl&#228;ndera, A., Schravenab, B., Bommhardt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD45+in+human+physiology+and+clinical+medicine.">CD45 in human physiology and clinical medicine.</a> Immunology Letters. 196, 22-32 (2018).</li><li>Hua, 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=Single-cell+RNA+sequencing+to+dissect+the+immunological+network+of+autoimmune+myocarditis.">Single-cell RNA sequencing to dissect the immunological network of autoimmune myocarditis.</a> Circulation. 142 (4), 384-400 (2020).</li><li>Grindberg, R. 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=RNA-sequencing+from+single+nuclei.">RNA-sequencing from single nuclei.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (49), 19802-19807 (2013).</li><li>Vanden Brink, S. 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=Single-cell+sequencing+reveals+dissociation-induced+gene+expression+in+tissue+subpopulations.">Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations.</a> Nature Methods. 14, 935-936 (2017).</li></ol>

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 concentrati...

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 ...

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 ...

Our protocol ensures both the cell viability and isolation efficiency of single cell suspension at relatively low costs. The relatively low costs and requirements for laboratory equipment are the main advantages of this technique. Single cell sequencing reveals the heterogeneity of non cardiomyocytes in cardiac pathophysiology, and also provides ideas for basic and clinical studies for heart disease.This protocol can be used in this studies on the post-MI cardiovascular environment such as the involvement of immune cells. For first time preparations, I suggest extra attention should be paid to the sample pepsin and digestion time. Once the buffers and solutions are prepared as described in the manuscript, place a euthanized mouse that had a myocardial infarction two weeks earlier in a supine position on the surgical table.Fix its hind limbs using adhesive tape. To excise the heart, spray the body with 70%ethanol. Make a vertical incision through the abdominal skin and muscles while avoiding the liver.Without puncturing the heart, carefully open the thorax and profuse the left ventricle with pre-cool PBS solution until the perfusion fluid runs colorless. Using forceps, gently lift the heart and cut away the excess tissues attached to its exterior with ophthalmic scissors. Place the heart in one milliliter of pre-cooled PBS in a 1.5 milliliter microcentrifuge tube.Transfer the heart into a well of a 24 well plate placed on ice. Pipet 150 microliters of the cardiac dissociation buffer into the well, and cut the heart into one millimeter sized pieces using scissors. Transfer the minced heart tissue into a 15 milliliter centrifuge tube containing five milliliters of the tissue dissociation buffer.Place the tube in a thermostatic reciprocating shaker. Pipet the suspension up and down 10 times every 20 minutes. To remove clumps and undigested tissue, filter the cell suspension through a 70 micrometer cell strainer using 25 milliliters of cell wash buffer.Rinse the centrifuge tube and the cell strainer. Next, filter the cell suspension through a 40 micrometer cell strainer to remove further cell clumps. Centrifuge the filtered cell suspension at 300 x g for five minutes at four degrees Celsius.Resuspend the cell sample in five volumes of RBC lysis buffer and incubate for five minutes at room temperature. Now, add four volumes of the cell wash buffer in cell suspension and centrifuge as demonstrated previously. After discarding the supernatant, resuspend the cells in 10 milliliters of the cell wash buffer and centrifuge once again.At the end of centrifugation, discard the supernatant and resuspend the pellet in one milliliter of cell wash buffer. Take 18 microliters of the suspension in another centrifuge tube and add two microliters of acridine orange propidium iodide staining solution. Add 10 microliter of this stained solution to a counting chamber slide.Evaluate the cell numbers and viability using an automatic cell counter. Cell fragments were observed in the single cell cardiac suspension. Fluorescence activated cell sorting effectively reduced the proportion of the cell fragments and cell clumping resulting in decreased average cell size.RNA sequencing proved an obvious separation of seven types of immune cells as demonstrated by the unsupervised clustering and reduced T-distributed stochastic neighbor embedding. Each immune cell showed a high expression of classic markers. Heart tissue needs to be cut small enough to avoid incomplete digestion, which leads to lots of cell lumps in the cell suspension.The single cell suspension obtained by this protocol can be further applied in flow analysis of primary cell culture. This protocol benefits the studies on the microenvironment of myocardial infection, which may help researchers to look beyond the cardiac tissues and consider myocardial infection as a systematic problem.

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1.8K 조회수.  Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province. 당사의 프로토콜은 상대적으로 저렴한 비용으로 단일 세포 현탁액의 세포 생존력과 분리 효율성을 모두 보장합니다. 실험실 장비에 대한 상대적으로 저렴한 비용과 요구 사항이이 기술의 주요 장점입니다. 단일 세포 시퀀싱은 심장 병태생리학에서 비심근 세포의 이질성을 밝히고 심장 질환에 대한 기본 및 임상 연구에 대한 아이디어를 제공합니다.이 프로토콜은 면역 ...