Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Summary

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Abstract

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.

The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.

This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Protocol

1. Reagent Preparation

1. The relaxation and preservation solution (RP Solution) is prepared as previously described with minor modifications¹. Briefly, the RP Solution consists of 2.77mM CaK₂EGTA, 7.23mM K₂EGTA, 20mM imidazole, 0.5mM dithiothreitol, 20mM taurine, 50mM K-MES, 6.56 MgCl₂, 5.7mM ATP, 14.3mM phosphocreatine, pH 7.1, adjusted at room temperature (RT). Filter the solution through a 0.45-μm filter to sterilize. Divide into 15 mL portions (Falcon polypropylene tubes) and store at -20 °C See discussion for recipes.
2. The Mitochondrial Respirometry Solution (MiR05) is prepared as previously describ....

Discussion

The saponin-permeabilized cardiac fiber technique offers a unique compromise between in vitro and in vivo assessment of mitochondrial OXPHOS oxygen consumption. Advantages of this technique include increased physiological relevance in comparison to isolated mitochondria as cellular architecture is preserved. While the plasma membrane is degraded, intracellular membrane structures including mitochondria^{12, 14}, sarcoplasmic reticulum¹⁴, myofilaments and the cytoskeleton ^{1, 17<.......}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100% Ethanol	Fisher Scientific	HC600
70% Ethanol	Fisher Scientific	HC-1000
Adenosine 5′-diphosphate monopotassium salt dihydrate (ADP)	Sigma-Aldrich	A5285
Albumin from bovine serum essentially fatty acid–free	Sigma-Aldrich	A-6003
Antimycin A	Sigma-Aldrich	A8674
Ascobic acid	Sigma-Aldrich	A4403
Adenosine 5′-triphosphate disodium salt hydrate (ATP)	Sigma-Aldrich	A2383
Atractyloside	Sigma-Aldrich	A6882
Calcium carbonate	Sigma-Aldrich	C4830
Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)	Sigma-Aldrich	C2920
Cytochrome c	Sigma-Aldrich	C7752
Digitonin	Sigma-Aldrich	D141
Dithiothreitol	Sigma-Aldrich	D9779
Ethylene glycol-bis-(2-amin–thylether)-N,N,N′,N′-tetraacetic acid (EGTA)	Sigma-Aldrich	E4378
Glutamic acid	Sigma-Aldrich	27647
HEPES	Sigma-Aldrich	H4034
Imidazole	Sigma-Aldrich	I5513
Ketamine	Pfizer Pharma GmbH		Ketaset
Lactobionic acid	Sigma-Aldrich	153516
Magnesium chloride (MgCl2)	Sigma-Aldrich	M9272
Magnesium chloride hexahydrate (MgCl2∙6H2O)	Sigma-Aldrich	M9272
Malic acid	Sigma-Aldrich	M1000
MES	Sigma-Aldrich	M3671
N,N,N’,N’-Tetramethyl- pphenylenediamine Dihydrochloride (TMPD)	Sigma-Aldrich	T3134
Oligomycin	Sigma-Aldrich	O4876
Phosphocreatine	Sigma-Aldrich	P7936
Potassium Chloride	Sigma-Aldrich	P9541
Potassium Hydroxide	Sigma-Aldrich	P5958
Potassium cyanide	Fluka	60178
Potassium phosphate monobasic	Sigma-Aldrich	P5655
Rotenone	Sigma-Aldrich	R8875
Saponin	Sigma-Aldrich	47036
Sodium Pentobarbital	Ceva Sante Animale	1715 138	Conc. 54.7 mg/ml
Sodium pyruvate	Sigma-Aldrich	P2256
Succinic acid	Sigma-Aldrich	S3674
Sucrose	Sigma-Aldrich	S7903
Taurine	Sigma-Aldrich	T8691
Xylazine	Bayer AG		Rompun
ddH2O
Ice
Oroboros Oxygraph-2k	Oroboros Instruments
Kimwipes	VWR international	21905-026
15ml polypropylene centrifuge tubes	VWR international	89004-368
50ml polypropylene centrifuge tubes	VWR international	89004-364
Straight Jewelers Forceps	George Tiemann & Co.	160-50B
Curved Jewelers Forceps	George Tiemann & Co.	160-57B
Straight Surgery Scissors	George Tiemann & Co.	105-402
Sterile Surgical Blade	VWR international	BD371610
0.45-μm Syringe filters	VWR international	CA28145-485
pH meter	VWR international	CA11388-308
Glass Petri dishes	VWR international	89000-300
12-well Polystyrene Tissue Culture Plates	VWR international	82050-926
Plate Stirrer	VWR international	97042-594
Fisherbrand Microbars	Fisher Scientific	14-511-67
Weigh Scale	VWR international	CA11278-162
10μl Hamilton Micro Syringe	Fisher Scientific	14-815-1
25μl Hamilton Micro Syringe	Fisher Scientific	14-824-7
50μl Hamilton Micro Syringe	Fisher Scientific	14-824-5
Nalgene Squeeze Bottles	Wilkem Scientific	LNA2407-1000
Polystyrene Weighing Dishes	VWR international	89106-750
Dissecting Microscope	Olympus Corporation