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.

Despite numerous studies, our understanding of the mitochondrial function regulation, specifically in vivo and under physiologically relevant conditions, is limited. One of the reasons is that majority of the methods developed for evaluating mitochondrial function rely on in vitro or ex vivo approaches, such as monitoring the oxygen consumption of isolated mitochondria supplemented with artificial substrates, and the indirect determination of mitochondrial function through morphology (e.g. electron microscopy), enzyme activity (e.g. aconitase activity), or intracellular ATP levels^9-11.

Recently, small molecule fluorescent indicators with relative mitochondrial enrichment have been applied to provide a glimpse of the mitochondrial signals, including membrane potential, calcium and reactive oxygen species (ROS), in intact cells^11-13. Moreover, several green fluorescent protein (GFP) based redox and ROS indicators have been developed to achieve more specific evaluation of the compartmentalized intracellular redox or ROS signals^14-16. Among this, we developed a genetically encoded superoxide indicator, the circular permuted yellow fluorescent protein, and targeted it into mitochondria (mt-cpYFP)¹⁷. mt-cpYFP can be excited at 405 or 488 nm with both emission peaks at 515 nm. The emission at 488 nm excitation is specifically responsive to superoxide as shown by previous in vitro and in vivo calibrations^17,18. The emission at 405 nm excitation is used as internal control (please refer to Figure 1 of Ref 17 for detailed information on the emission and excitation spectra of mt-cpYFP under various conditions). With time-lapse confocal imaging, this indicator detects bursting superoxide production events, named superoxide flashes, in single mitochondria of intact cells. Superoxide flash serves as a composite function of mitochondrial respiration, accompanying transient mitochondrial membrane depolarization and ROS production^17-20. Recently, we have generated the pan-tissue mt-cpYFP transgenic mice using the pUC-CAGGS-mt-cpYFP vector^17,19 on C57/BL6 background and verified the strong expression of this indicator in the heart, skeletal muscles and other tissues (Figure 2). The transgenic mice will be available for interested academic investigators upon request and MTA approval by the University of Washington.

In this study, we describe in situ imaging of superoxide flashes in Langendorff perfused heart as well as in vivo imaging of flash events in skeletal muscles of anesthetized mt-cpYFP transgenic mice^17,19. This technology allows real time monitoring of single mitochondrial ROS production events in a physiologically relevant condition or in vivo ^21,22. It is also feasible to use the system to monitor other single mitochondrial parameters such as membrane potential and calcium with appropriate fluorescent indicators. Further, simultaneous or parallel evaluation of mitochondrial function with intracellular events (e.g. calcium transients) or heart function (e.g. ejection fraction) can be achieved. Pathological perturbations, such as ischemia and reperfusion, can be applied to the perfused heart to assess the impact of stress on single mitochondrial function in the intact myocardium.

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 sterilizing the clamp, scissors, and forceps in 70% ethanol and then rinsing in sterilized ddH₂O.
5. Turn on the heart perfusion system, adjust the temperature on the water bath and circulator, set the speed of peristaltic pump to 6 ml/min.
6. Bubble the KHB with 95% O₂/5% CO₂ gas, and monitor the flow rate and temperature (37 °C, by a fiber optic temperature probe) of the effluent. The system needs about 20 min for gas and temperature equilibration.

2. Confocal Imaging of Skeletal Muscles In Vivo

1. Anesthetize mouse with pentobarbital (80 mg/kg, i.p.). The animal will reach surgical anesthesia (no response to toe pinch) within 10-15 min and remain in this status for 1-1.5 hr, sufficient for the in vivo imaging of skeletal muscles.
2. Remove hairs on one of the hindlimbs and sterilize the skin with 70% ethanol.
3. Make an incision on the skin along the outer side of the limb to expose the gastrocnemius muscles.
4. Pick up the epimysium gently with a sharp forceps and make an incision through it with scissors. Further dissect to remove the epimysium and expose the muscle fibers beneath it.
5. For in vivo loading of TMRM into the skeletal muscle, include TMRM (500 nM) in the isotonic balanced salt solution to immerse muscle for 30 min and then wash out by indicator-free solution.
6. Put the mouse on its side on the confocal microscope (Zeiss LSM 510) stage and restrain the rear limb in a position that the exposed skeletal muscle is facing against the coverslip that forms the bottom of the chamber. The coverslip is in between the muscle tissue and the inverted objective (40X oil).
7. Press the leg down gently to make tight contact between the tissue and the coverslip. Immerse the exposed muscles in isotonic balanced salt solution.
8. Record two dimensional (2D, xy) confocal images at a sampling rate of one second per frame. The intensity of each pixel is digitized at 8-bit depth. Usually, a serial scan contains 100 frames.
9. Collect sequential images by first exciting at 405 nm and collecting at >505 nm then at 488 nm while collecting at >505 nm for dual wavelength excitation imaging of mt-cpYFP. Use sequential excitation at 405, 488 and 543 nm, and collect emission at 505-545, 505-545, and >560 nm, respectively, for triple wavelength excitation imaging of mt-cpYFP and TMRM.

3. Confocal Imaging of Perfused Mouse Heart

1. Immediately after the in vivo imaging of skeletal muscles, inject the mouse with 200 units of heparin (i.p.). Ten minutes later, euthanize the mouse by thoracotomy and quickly remove the heart with lungs and thymus attached to it.
2. Quickly remove the lung in ice-cold buffer. Identify the lobes of the thymus and gently peel back to expose the ascending aorta.
3. Remove the thymus and isolate the aorta by carefully removing any surrounding tissue.
4. Make a cut at the upper end of ascending aorta before the first branch of aortic arch.
5. Hold the wall of the aorta gently with two micro suturing forceps to expose the lumen and carefully place the aorta onto the cannula (PE50 tube). The aorta is held in place with a micro vessel clamp while sutures are quickly tied around the aorta.
6. Remove the clamp, carefully check the cannula with forceps to make sure the tip of the cannula is above aortic root. Additional ties are added as necessary to hold the heart in place.
7. Turn on the peristaltic pump and perfuse the heart at 1 ml/min. The heart will resume beating upon perfusion.
8. Adjust the position of the perfusion system and put the heart on the microscope stage. The stage has an adaptor that allows heating of the chamber that holds the heart.
9. Add 1 ml of KHB perfusion solution in the chamber to partially submerge the heart. Monitor temperature of the chamber at 37 °C. Remove effluent from the chamber by using a peristaltic pump.
10. Increase the speed of peristaltic pump gradually to provide sufficient flow (approximately 2 ml/min) to the heart.
11. After 10 min of stabilization, perfuse the heart with 10 μM blebbistatin and 100-500 nM TMRM. Heart beat will slow down after 10 min.
12. Apply gentle pressure on the heart to ensure tight contact of the heart with the coverslip at the bottom of the chamber and to further suppress heartbeat.
13. Follow the same procedure for confocal imaging of intact myocardium as described in step 2.8 above. Carefully adjust the focal plane to reveal the clearest image possible.

4. Image Processing and Data Analysis

1. Use tools provided by the "Physiological Analysis" module of the confocal software to analyze single mitochondrial flashes and membrane potential changes. This module is often included in the image acquiring software of the confocal system and allows determination of certain areas in the image as well as output of fluorescence intensity with time labels.
2. Open the database and then the serial 2D image file to be analyzed.
3. Click the "Region of Interest (ROI) mean" to switch to the "mean of ROIs" mode. Click the "Region of Interest (ROI) mean" to switch to the "mean of ROIs" mode.
4. Turn off the display of other channels except the channel of cpYFP 488 nm for selecting the flashes.
5. Zoom in the image and manually move slide bar to play the serial 2D images.
6. Identify single mitochondrial superoxide flashes by locating the site where fluorescent signal increases transiently. Use the appropriate ROI tool to mark the flashes. The trace showing time-dependent fluorescence change of each ROI will show up beside the image.
7. After selecting all the flashes, turn on the display of other channels. Select an ROI on the image outside of the cell for background signal subtraction. Output the average fluorescence of each ROI together with the time labels.
8. Record the number of flashes in each of the serial scanning image file together with the scan time and area of the cell. Use Excel to calculate flash frequency as number of flashes per 100 sec/1,000 μm² cell area.
9. Use a custom-developed program written in Interactive Data Language (IDL, ResearchSystems) to calculate the parameters of each flash (amplitude, time to peak and decay time). IDL software is commercially available and the custom-developed program for flash parameter analysis can be obtained from the author upon request.

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.