Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Summary

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Abstract

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-α 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-α 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

Introduction

Heart failure is the leading cause of deaths, and myocarditis is one predominant cause of heart failure in young adolescents¹. Most patients affected with myocarditis remain asymptomatic and the disease is spontaneously resolved². However, 10-20% of those affected can develop chronic disease, leading to dilated cardiomyopathy (DCM)³. Various animal models have been developed to study the immune pathogenesis of myocarditis. The disease can be induced in myocarditis-susceptible A/J and Balb/c mice by immunizing the animals with cardiac myosin heavy chain (Myhc)-α or its immunodominant peptide fragments or by infecting with pathogens....

Protocol

ETHICS STATEMENT:

All animal procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and approved by the University of Nebraska-Lincoln, Lincoln, NE.

1. Induction of Experimental Autoimmune Myocarditis

1. Prepare the peptide solution by dissolving Myhc-α 334-352 in 1x phosphate-buffered saline to a final concentration of 2 mg/1.5 ml.
2. Prepare the pertussis toxin (PT) by adding 1 ml of sterile 1x PBS to a vial containing 50 μg of lyophilized PT to obtain a stock concentration of 50 ng/μl. Take 20 μl of the stock i....

Results

In this report, the utility of MRM technique as a non-invasive modality to determine the structural and functional changes in the hearts of animals affected with EAM is shown. Myocarditis was induced in A/J mice by immunizing the animals with Myhc-α 334-352 in CFA⁷, and the animals were subjected to MRM experiments on day 21 post-immunization. The MRM imaging was performed on live animals under isoflurane anesthesia at 9.4 T (400 MHz for protons) using an 89 mm vertical-bore magnet equipped with triple axis gr.......

Discussion

This study describes the MRM procedure and its utility as a non-invasive tool to ascertain cardiac abnormalities in mice affected with autoimmune myocarditis. Since the histologic features of EAM resemble postinfectious myocarditis of humans, mouse models are commonly employed to delineate the immune mechanisms of myocardial injuries^23-25. However, the animals affected with myocarditis appear clinically normal, and the diagnosis is made based on histology at the termination of experiments⁷. The anim.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)	Neopeptide, Cambridge, MA		Store at 4 °C
CFA	Sigma Aldrich, St Louis, MO	5881	Store at 4 °C
MTB H37Rv extract	Difco Laboratories, Detroit, MI	231141	Store at 4 °C
PT	List Biologicals Laboratories, Campbell, CA	181	Store at 4 °C
1x PBS	Corning, Manassas, VA	21-040-CV	Store at 4 °C
Isoflurane	Piramal Healthcare, Mumbai, India	NDC66794-013-25
Female A/J mice	Jackson Laboratories, Bar Harbor, ME	646
Luer-lok sterile 1 ml syrringe	BD, Franklin Lakes, NJ	309628
Luer-lok sterile 3 ml syrringe	BD, Franklin Lakes, NJ	309657
Sterile needle, 18 G	BD, Franklin Lakes, NJ	305195
Sterile needle, 27 1/2 G	BD, Franklin Lakes, NJ	305109
3-way stopcock	Smiths Medical ASD, Inc. Dublin, OH	MX5311L
Kerlix gauze bandage rolls	Covidien, Mansfield, MA	6720
Kimwipes	Kimberly-Clark Professional, Roswell, GA	34155
Protouch Stockinette	Medline Industries, Mundelein, IL	30-1001
Sterile surgical scissors and forceps	INOX tool Corporation
Micro oven	GE Healthcare,
ThermiPAQ hot and cold therapy system	Theramics Corporation, Springfield, IL
Reptile heating lamp	Energy Savers Unlimited, Inc. Carson, CA
3M Transpore tapes	Target Corporation, MN
Up and Up Polymyxin B sulfate/Bacitracin/Neomycin sulfate antibiotic ointment	Target Corporation, MN
North Safety DeciDamp-2PVC foam ear plugs	North Safety Products, Smithfield, RI
Cotton tipped applicator, 6’’ wooden stem	Jorgensen Laboratories, Inc. Loveland, CO
Anesthesia induction chamber	Summit Anesthesia Solutions, Ann Harbor, MI
Summit Anesthesia Support system for regulating flow of anesthesia	Summit Anesthesia Solutions, Ann Harbor, MI
Specially designed animal holder	Agilent Technologies, Santa Clara, CA
Bickford Omnicon F/Air anesthesia gas filter unit	A.M. Bickford, Inc. Wales Center, NY
Pulse-oximeter module, MR compatible small animal monitoring and gating system	Small Animal Instruments, Inc. Stony Brook, NY
Oxygen cylinder	Matheson-Tri Gas, North-Central Zone, Lincoln, NE
Gas regulator	Western Medica, West Lake, OH
Signal breaking module, MR compatible small animal monitoring and gating system	Small Animal Instruments, Inc. Stony Brook, NY
9.4 T (400 MHz) 89 mm vertical core bore MR scanner	Agilent Technologies, Santa Clara, CA
4 cm millipede micro-imaging RF coil	Agilent Technologies, Santa Clara, CA
SAM PC monitor	Small Animal Instruments, Inc. Stony Brook, NY
Quantitative Medical Image analysis software	http://segment.heiberg.se;  Segment v1.8 R1430,  Medviso, Oresunds region, Sweden
Matlab software	The Mathworks, Inc.  Natick, MA
Computer-Unix operating system