Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Summary

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Abstract

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Introduction

Mitochondria play a central role in cell bioenergetics, free radical signaling, redox homeostasis, ion regulation, and cell fate determination^1,2. Mitochondria dysfunction often accompanies and underlies the pathogenesis of diseases^3-6. Especially in the muscle systems such as the heart and skeletal muscles, mitochondrial respiration provides the majority of ATP to support timely regulation of intracellular calcium and robust force development^7,8. These muscles possess a large number of mitochondria that often occupy up to 20-40% of the total cell volume and are "fixed" in between myofilaments².

Protocol

1. Experiment Preparation

1. Prepare the isotonic balanced salt solution (50 ml) containing: 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 2 mM MgCl₂ and 10 mM HEPES (pH 7.2) for in vivo skeletal muscle imaging.
2. Prepare 1 L of Krebs-Henseleit buffer (KHB) containing: 118 mM NaCl, 5.3 mM KCl, 1.2 mM MgSO₄, 0.5 mM EDTA and 25 mM NaHCO₃.
3. Bubble the KHB with gas containing 95% O₂/5% CO₂ for 10-15 min prior to the addition of 2 mM CaCl₂. Add metabolic substrates (e.g. 10 mM glucose and 0.5 mM pyruvate).
4. Prepare the surgical instruments by steril....

Results

According to this protocol, in vivo imaging of single mitochondrial events can be done in skeletal muscles of anesthetized mice followed by in situ imaging in perfused heart (Figure 1). The optimal setting of the imaging conditions will ensure clear images of the intact muscle tissues and with single mitochondrion resolution (Figure 2). TMRM is often used to verify the location of mt-cpYFP and should show a complete overlapping pattern with the mt-cpYFP signal (.......

Discussion

Imaging single mitochondrial events in live animal or perfused organs has significant advantage over traditional methods for mitochondrial function evaluation^{17,19,21,22,24,25}. The technique described here can achieve real-time in situ determination of mitochondrial function in a real physiological condition at the subcellular resolution. This is particularly useful, when combined with other measurements, to systemically study the role of mitochondria in the normal function of a particular organ or ce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
Blebbistatin	Toronto Research Chemicals	B592500
CaCl2	Acros Organics	AC34961-5000
EDTA	Fisher Scientific	BP120-500
D-Glucose	Sigma-Aldrich	G8270-1
HEPES	Sigma-Aldrich	H7006-500
KCl	Sigma-Aldrich	P9541-1
MgCl2•6H2O	Fisher Scientific	BP214-500
MgSO4•7H2O	Sigma-Aldrich	M1880-1
NaCl	Fisher Scientific	BP358-212
NaH2PO4	Sigma-Aldrich	S8282-500
NaHCO3	Sigma-Aldrich	S6014-1
Pyruvate	Sigma-Aldrich	P2256-25
TMRM	Invitrogen	T-668
EQUIPMENT
Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including "Physiological Analysis" module.