Name: analyzing oxygen consumption rate in primary cultured mouse neonatal cardiomyocytes using an extracellular flux analyzer
Uploaded: 2019-02-13T13:00:00
Description: Watch this Scientific Journal Video about Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer at JoVE.com

We would like to thank all Ross lab and Murphy lab members. This work is supported by American Heart Association (14SDG17790005) to Y.C. NIH (HL115933, HL127806) and VA Merit (BX003260) to R.S.R.

For work with neonatal mice, please refer to local university/institute guidelines set forth by the animal care programs and adhere to one&#8217;s institutional and other appropriate regulations. All methods described in this protocol have been approved by the UC San Diego Institutional Animal Care and Use Committee (IACUC) and adhere to federal and state regulations.
1. Preparation of Reagents
<ol>
	<li>Prepare 25 mL of pre-digestion solution: HBSS (without Ca2+ ...

<ol>
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In this study, we have established a simple protocol for isolating and culturing mouse neonatal cardiomyocytes. By using these cardiomyocytes, we also optimized the conditions to measure oxygen consumption rate by using an extracellular flux analyzer system. The protocol allows one to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the principal working cells of the heart, akin to what would be measured in the intact organ. Our protocol is different from ...

To sustain a continuous cardiac contractile function, cardiomyocytes must maintain a constant supply of cellular energy primarily in the form of ATP1. In the heart, approximately 95% of ATP is generated by mitochondria, mainly through oxidative phosphorylation, showing that mitochondria play a crucial bioenergetic role in cardiac function2,3. Supporting this notion is that dysregulation of mitochondrial function can lead to cardiomyopathy and heart failure4,5. Conversely, restoring mitochondrial function has been shown to improve cardiac function of the failing heart6,7. Therefore, studying the mechanism of mitochondrial bioenergetics and identifying novel regulators of mitochondrial function in cardiomyocytes will not only reveal mechanistic insights of cardiac energy production but also could provide insight that will lead to development of new therapeutic targets to treat heart diseases6,8.
Compared to the whole heart, which contains a mixture of myocytes and non-myocytes9, cardiomyocyte cultures are extremely pure, with minimal contamination of non-myocytes from the heart, such as fibroblasts and endothelial cells10. In addition, isolating cardiomyocytes from neonatal pups enables culturing a large number of cells in a small amount of time, compared to isolating cells from adult hearts10,11. Most importantly, primary cultured adult mouse cardiomyocytes have short survival times (e.g. 24 h) and at longer time points de-differentiate. Neonatal mouse cardiomyocytes can survive and be manipulated for upwards of 7 days in culture, making them ideal for testing the effects of drug compounds and gene manipulation on the function of mitochondria in cardiomyocytes10. Of course, there are significant biological differences between the adult and neonatal cells, but the longer duration available for culture of neonatal cells makes them appropriate for many different types of studies, including those of mitochondrial function.
To date, primary cultured neonatal mouse and rat cardiomyocytes have been used as models to study cardiac bioenergetics12,13. In recent years, studies used an extracellular flux analyzer to measure oxygen consumption rate (OCR) and evaluate oxidative capacity in mouse and rat neonatal cardiomyocytes14,15. While compared to rats, the cell viability of mouse neonatal cardiomyocytes is lower and has greater variability16. Also, the ability to study cells from genetically engineered mouse models makes the mouse cell model very important. Given that OCR studies are so sensitive to cell number and seeding density, development of a reproducible, reliable, and simple protocol to achieve consistent cell yield and viability is needed.
Here, we report an optimized protocol that has been developed which uses cultured mouse neonatal cardiomyocytes along with a 96-well-format extracellular flux analyzer for OCR analysis. This protocol greatly increases reproducibility of the assay. In addition, the protocol not only provides a novel and reproducible method for OCR analysis, but also could be adapted to a larger size culture for other experimental purposes, such as that which may be needed to study myofibrillar functions and intracellular signaling pathways.
In particular, this protocol describes a one-day procedure for isolation and culture of neonatal mouse cardiomyocytes in a 96-well cell culture plate. In addition, it describes the procedure to measure oxygen consumption using an extracellular flux analyzer. All solutions used are sterile or sterile filtered. All tools are sterilized by 75% ethanol. We provide a&#160;Table of Materials for various parts of the procedure. For culturing cardiomyocytes, all procedures and steps are performed in a standard cell culture hood. This protocol is developed for the isolation of neonatal mouse hearts from one litter (approximately 8-10 pups). However, the protocol can also be adapted for isolating cardiomyocytes from multiple litters.

<table border="0" cellpadding="0" cellspacing="0" style="width:924px;" width="924">
	<tbody>
		<tr height="28">
			<td height="28" style="height:28px;width:269px;">Antimycin A</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">A8674</td>
			<td style="width:288px;">Inhibits complex III of the mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Cell strainer 100 &#956;m pores</td>
			<td style="width:187px;">FALCON</td>
			<td style="width:180px;">352360</td>
			<td style="width:288px;">To capture undigested tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Collagenase type II</td>
			<td style="width:187px;">Worthington</td>
			<td style="width:180px;">LS004176</td>
			<td style="width:288px;">To make collagenase digestion solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">D-Glucose</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">75351</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM high glucose</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">11965-092</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">DMEM without NaHCO3, Glucose, pyruvate, glumanine, and Hepes</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">D5030-10X1L</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">C2920</td>
			<td style="width:288px;">Uncouples mitochondrial respiration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fetal bovine serum (FBS)</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">26140-079</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Fibronectin from bovine plasma</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">F1141-5MG</td>
			<td style="width:288px;">To make coating solution for tissue culture plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fine scissors</td>
			<td style="width:187px;">Fine Sciences Tools</td>
			<td style="width:180px;">14060-10</td>
			<td style="width:288px;">For dissection of hearts</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Gelatin from porcine skin</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">G-1890</td>
			<td style="width:288px;">To make coating solution for tissue culture plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">HBSS (Hank&#39;s balnced salt solution,without Ca2+, Mg2+)</td>
			<td style="width:187px;">Cellgro</td>
			<td style="width:180px;">21-022-CV</td>
			<td style="width:288px;">To wash hearts and make pre-digestion and collagnase digestion solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">HEPES (1M)</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">15630080</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Horse serum</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">26050-088</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">L-Glutamine</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">G-3126</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">M-199</td>
			<td style="width:187px;">Cellgro</td>
			<td style="width:180px;">10-060-CV</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:269px;">Moria spoon</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">NC9190356</td>
			<td style="width:288px;">To wash hearts&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Oligomycin</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">75351-5MG</td>
			<td style="width:288px;">Inhibits mitochondrial ATP synthase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">RIPA buffer</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">89900</td>
			<td style="width:288px;">To lyse the cells for protein assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Rotenone</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">R8875</td>
			<td style="width:288px;">Inhibits complex I of the mitochondria</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Seahorse XFe96 Extracellular Flux 
			Analyzer</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:180px;"></td>
			<td style="width:288px;">Device used to analyze oxygen consumption rate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Seahorse XFe96 FluxPak</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:180px;">102601-100</td>
			<td style="width:288px;">Package of flux analyzer culture plates, sensor cartridges, and calibrant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Sodium pyruvate</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">P2256</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Straight scissors</td>
			<td style="width:187px;">Fine Sciences Tools</td>
			<td style="width:180px;">91401-12</td>
			<td style="width:288px;">For dissection of hearts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe filter 0.2 &#956;m size</td>
			<td style="width:187px;"></td>
			<td style="width:180px;"></td>
			<td style="width:288px;">For sterile filtration of digestion medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Trypsin</td>
			<td style="width:187px;">USB Corporation</td>
			<td style="width:180px;">22715 25GM</td>
			<td style="width:288px;">To make pre-digestion solution</td>
		</tr>
	</tbody>
</table>

analyzing oxygen consumption rate in primary cultured mouse neonatal cardiomyocytes using an extracellular flux analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

By using the protocol described, hearts were isolated from day 0 neonatal pups. 5 x 105 cells/pup were obtained, and cardiomyocytes were seeded at densities of 10 x 103, 20 x 103, or 30&#160;x 103 cells/well, in 96 well plates (Figure 2A). After overnight culture, cardiomyocytes were found well-attached to the coated plastic surface and there were very few unattached cells (the unattached cells will still appear as ...

Watch this Scientific Journal Video about Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer at JoVE.com

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Research

JoVE Journal

Medicine

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

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Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

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Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

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Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

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Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...