Ratiometric imaging of calcium during ischemia-reperfusion injury in isolated mouse hearts using Fura-2

Venkataraman Raghav, Holcomb, Harder Rene, Knollmann, Baudenbacher, Baudenbacher Franz

Description

We present an easily implementable method for measuring Fura-2 fluorescence from isolated mouse hearts using a commercially available switching light source and CCD camera. After calibration, it provides a good estimate of intracellular [Ca 2+ ] with both high spatial and temporal resolutions, permitting study of changes in dispersion of diastolic [Ca 2+ ], Ca 2+ transient dynamics, and conduction velocities in mouse hearts. In a proof-of-principle study, we imaged isolated Langendorff-perfused mouse hearts with reversible regional myocardial infarctions. Methods Isolated mouse hearts were perfused in the Landendorff-mode and loaded with Fura-2. Hearts were then paced rapidly and subjected to 15 minutes of regional ischemia by ligation of the left anterior descending coronary artery, following which the ligation was removed to allow reperfusion for 15 minutes. Fura-2 fluorescence was recorded at regular intervals using a high-speed CCD camera. The two wavelengths of excitation light were interleaved at a rate of 1 KHz with a computer controlled switching light source to illuminate the heart. Results Fura-2 produced consistent Ca 2+ transients from different hearts. Ligating the coronary artery rapidly generated a well defined region with a dramatic rise in diastolic Ca 2+ without a significant change in transient amplitude; Ca 2+ handling normalized during reperfusion. Conduction velocity was reduced by around 50% during ischemia, and did not recover significantly when monitored for 15 minutes following reperfusion. Conclusions Our method of imaging Fura-2 from isolated whole hearts is capable of detecting pathological changes in intracellular Ca 2+ levels in cardiac tissue. The persistent change in the conduction velocities indicates that changes to tissue connectivity rather than altered intracellular Ca 2+ handling may be underlying the electrical instabilities commonly seen in patients following a myocardial infarction.

Sujets

• Optical mapping
• Nerve conduction study
• Reperfusion injury

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 30
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Venkataramanet al. BioMedical Engineering OnLine2012,11:39 http://www.biomedicalengineeringonline.com/content/11/1/39

R E S E A R C HOpen Access Ratiometric imaging of calcium during ischemiareperfusion injury in isolated mouse hearts using Fura2 1 2,53 41* Raghav Venkataraman , Mark R Holcomb, Rene Harder , Björn C Knollmannand Franz Baudenbacher

* Correspondence: [e-mail] 1 Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA Full list of author information is available at the end of the article

Abstract Background:We present an easily implementable method for measuring Fura2 fluorescence from isolated mouse hearts using a commercially available switching light source and CCD camera. After calibration, it provides a good estimate of 2+ intracellular [Ca] with both high spatial and temporal resolutions, permitting study 2+ 2+ of changes in dispersion of diastolic [Ca], Catransient dynamics, and conduction velocities in mouse hearts. In a proofofprinciple study, we imaged isolated Langendorffperfused mouse hearts with reversible regional myocardial infarctions. Methods:Isolated mouse hearts were perfused in the Landendorffmode and loaded with Fura2. Hearts were then paced rapidly and subjected to 15 minutes of regional ischemia by ligation of the left anterior descending coronary artery, following which the ligation was removed to allow reperfusion for 15 minutes. Fura 2 fluorescence was recorded at regular intervals using a highspeed CCD camera. The two wavelengths of excitation light were interleaved at a rate of 1 KHz with a computer controlled switching light source to illuminate the heart. 2+ Results:Fura2 produced consistent Catransients from different hearts. Ligating the coronary artery rapidly generated a well defined region with a dramatic rise in 2+ 2+ diastolic Cawithout a significant change in transient amplitude; Cahandling normalized during reperfusion. Conduction velocity was reduced by around 50% during ischemia, and did not recover significantly when monitored for 15 minutes following reperfusion. Conclusions:Our method of imaging Fura2 from isolated whole hearts is capable 2+ of detecting pathological changes in intracellular Calevels in cardiac tissue. The persistent change in the conduction velocities indicates that changes to tissue 2+ connectivity rather than altered intracellular Cahandling may be underlying the electrical instabilities commonly seen in patients following a myocardial infarction. Keywords:Optical Mapping, Langendorff Mouse Heart, WholeHeart Fura2, IschemiaReperfusion, Conduction Velocity, Reperfusion Injury, LAD ligation, Ratiometric Calcium Imaging

Background Coronary heart disease affects approximately 1 in 8 adults and causes 1 in 6 deaths in the United States [1]. Occlusion of the coronary arteries can result in a myocardial in farction (MI) wherein affected cardiac tissue does not receive sufficient oxygenated

© 2012 Venkataraman et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.