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

Summary

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.

Abstract

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.

Introduction

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 function^2,3. Supporting this notion is that dysregulation of mitochondrial function can lead to cardiomyopathy and heart failure^4,5. Conversely, restoring mitochondrial function has been shown to improv....

Protocol

For work with neonatal mice, please refer to local university/institute guidelines set forth by the animal care programs and adhere to one’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

1. Prepare 25 mL of pre-digestion solution: HBSS (without Ca²⁺ .......

Discussion

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 .......

Materials

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