Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

Summary

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

Abstract

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Introduction

The goal of this protocol was to provide detailed steps to obtain mitochondria-enriched preparation from previously frozen heart tissue with a new technology of low energy mechanical disruption of tissue that improves tissue lysis and extraction of mitochondria. With this method, improved extraction efficiency without generating high shear stress or high temperature and short homogenization time (10-12 s) become achievable¹.

To obtain mitochondria from archived frozen tissue is a valuable asset to perform both functional² and biochemical studies³ otherwise not easily re....

Protocol

Frozen offspring heart tissues were received from Dr. Sean Newcomer along with the institutional IACUC approval letter. The heart tissues were obtained from a long-term longitudinal study, flash-frozen in liquid nitrogen, and stored at -80 °C for future use. All protocols concerning the processing of offspring heart tissue followed the guidelines of Kansas City University IBC and IACUC committees.

1. Preparation of buffers and reagents

NOTE: Prepare all samp.......

Discussion

The critical steps for this protocol are indicated here. First, tissue homogenization should be carefully handled so that excessive sheer effects will not be applied during the tissue homogenization process. A tissue shredder should be used, which is a part of pressure cycling technology (PCT) for initial tissue homogenization⁹. This step will reduce the excessive stroke cycle of glass-on-glass homogenizer (Figure 1B) that may further destroy already fragile mitochond.......

Materials

Name	Company	Catalog Number	Comments
Amino caproic acid	Sigma/Aldrich	A2504-100G
Anti-Hu Total OxPhos complex kit	Invitrogen	458199
anti-VDAC antibody	abcam	ab15895	use 1 µg/mL
Coomassie G-250	ThermoSientific	20279
Coomassie GelCode Blue	ThermoScientific	24592
Digitonin	Cabiochem	300410
Glass-Glass pestle homogenizer	VWR	KT885451-0020
Image Studio	LICOR
IR-Dye conjugated anti-Rabbit Ab	LICOR	LC0725
Multiwell plate reader	BioTek	Synergy HT
Native molecular weight marker	ThermoFisher	BN2001
Nylon mesh monofilament	Small Part Inc	CMN-74
Orbital shaker	ThermoScientfic
PCT Shredder	Pressure Bioscience Inc
SEA BLOCK Blocking buffer	ThermoScienctific	37527
Shredder PULSE Tube	Pressure Bioscience Inc	FT500-PS
Table top centrifuge	Eppendorf	5418
Trypsin	Amresco	M150-1G
Trypsin inhibitor	Amresco	M191-1G	Requires fresh preparation