Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

Summary

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

Abstract

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to investigate metabolic capability. To obtain the measures of mitochondrial respiration, fresh biological samples must be used, and the entire laboratory procedure must be completed within approximately 2 h. Furthermore, multiple pieces of specialized equipment are required to perform these laboratory assays. This creates a challenge for measuring mitochondrial respiration in the tissues of wild animals living far from physiology laboratories as live tissue cannot be preserved for very long after collection in the field. Moreover, transporting live animals over long distances induces stress, which can alter mitochondrial energetics.

This manuscript introduces the Auburn University (AU) MitoMobile, a mobile mitochondrial physiology laboratory that can be taken into the field and used on-site to measure mitochondrial metabolism in tissues collected from wild animals. The basic features of the mobile laboratory and the step-by-step methods for measuring isolated mitochondrial respiration rates are presented. Additionally, the data presented validate the success of outfitting the mobile mitochondrial physiology laboratory and making mitochondrial respiration measurements. The novelty of the mobile laboratory lies in the ability to drive to the field and perform mitochondrial measurements on the tissues of animals captured on site.

Introduction

To date, studies designed to measure mitochondrial energetics have been limited to laboratory animals or animals captured near established physiology laboratories, which precluded scientists from performing mitochondrial bioenergetic studies in tissues collected from animals during such activities as migration, diving, and hibernation^1,2,3,4,5,6. While many investigators have successfully measured the basal and peak metabolic rates and daily energy expenditures of wild ani....

Protocol

The following sections describe the mitochondrial laboratory methods. All animal handling and tissue collection procedures were approved by the Auburn University Institutional Animal Care and Use Committee (#2019-3582).

1. Description of buffers used for data collection

NOTE: These buffers can be prepared in a stationary laboratory and moved to the mobile laboratory prior to the field trip (unless otherwise noted below).

1. Prepare the skeletal muscle mitochondrial isolation buffer with bovine serum albumin (BSA), as seen in Table 1.
   1. Dissolve chemicals in deionized water (~ 9....

Results

The current manuscript investigated the mitochondrial respiration of wild-derived Mus musculus (n = 7, male = 5, female = 2; age = 1.30 ± 0.2 years) in a mobile mitochondrial physiology laboratory (Figure 1). To measure skeletal muscle mitochondrial respiration, the entire hindlimb, thus aerobic and anaerobic muscle, was used for mitochondrial isolation (Figure 2). Examples of raw mitochondrial respiration data are shown in Fig.......

Discussion

The mobile mitochondrial physiology laboratory enables researchers to isolate mitochondria and measure mitochondrial respiration rates within 2 h of tissue collection at remote field sites. The results presented herein suggest that measurements of mitochondrial respiration made in the AU MitoMobile are comparable to measurements made in a university research laboratory. Specifically, the values for state 3, state 4, and RCR for wild-derived Mus musculus presented here are comparable with previously published res.......

Materials

Name	Company	Catalog Number	Comments
1.7 mL centrifuge tubes	VWR	87003-294
2.0 mL centrifuge tubes	VWR	87003-298
50 mL centrifuge tubes	VWR	21009-681	Nalgene Oak Ridge Centrifuge Tube
ADP	VWR	97061-104
ATP	VWR	700009-070
Bradford	VWR	7065-020
Clear 96 well plate	VWR	82050-760	Greiner Bio-One
Dounce homogenizer	VWR	22877-284	Corning
EGTA	VWR	EM-4100
Filter paper			Included with Hansatech OxyGraph
Free-fatty acid BSA	VWR	89423-672
Glucose	VWR	BDH8005-500G
Glutamate	VWR	A12919
Hamilton Syringes	VWR	60373-985	Gaslight 1700 Series Syringes
Hansatech OxyGraph	Hansatech Instruments Ltd		No Catalog Number, but can be found under Products --> Electrode Control Units
KH2PO4	VWR	97062-350
Malate	VWR	97062-140
Mannitol	VWR	97061-052
Membrane			Included with Hansatech OxyGraph
MgCl2	VWR	97063-152
MOPS	VWR	80503-004
Policeman	VWR	470104-462
Polytron	Thomas Scientific	11090044
Potassium chloride (KCl)	VWR	97061-566
Protease	VWR	97062-366	Trypsin is commonly used; however, other proteases can be used.
Pyruvic acid	VWR	97061-448
Sodium Dithionite	VWR	AA33381-22
Succinate	VWR	89230-086
Sucrose	VWR	BDH0308-500G
Tris-Base	VWR	97061-794
Tris-HCl	VWR	97061-258