Autoimmune Myocarditis, Valvulitis, and Cardiomyopathy

Jennifer M. Myers1, DeLisa Fairweather2, Sally A. Huber3, Madeleine W. Cunningham1

1 University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 2 Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, 3 University of Vermont, Colchester, Vermont
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.14
DOI:  10.1002/0471142735.im1514s101
Online Posting Date:  April, 2013
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Abstract

Myocarditis and valvulitis are inflammatory diseases affecting myocardium and valve. Myocarditis, a viral‐induced disease of myocardium, may lead to dilated cardiomyopathy and loss of heart function. Valvulitis leads to deformed heart valves and altered blood flow in rheumatic heart disease. Animal models recapitulating these diseases are important in understanding the human condition. Cardiac myosin is a major autoantigen in heart, and antibodies and T cells to cardiac myosin are evident in inflammatory heart diseases. This unit is a practical guide to induction and evaluation of experimental autoimmune myocarditis (EAM) in several mouse strains and the Lewis rat. Purification protocols for cardiac myosin and protocols for induction of EAM by cardiac myosin and its myocarditis‐producing peptides, and coxsackievirus CVB3, are defined. Protocols for assessment of myocarditis and valvulitis in humans and animal models provide methods to define functional autoantibodies targeting cardiac myosin, β‐adrenergic, and muscarinic receptors, and their deposition in tissues. Curr. Protoc. Immunol. 101:15.14.1‐15.14.51. © 2013 by John Wiley & Sons, Inc.

Keywords: myocarditis; rheumatic fever; autoimmunity; coxsackievirus; cardiac myosin

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Induction of EAM in Mice by Active Immunization with Cardiac Myosin
  • Alternate Protocol 1: Induction of EAM in Mice by Adoptive Transfer of Cardiac Myosin–Stimulated T Cells
  • Basic Protocol 2: Induction of EAM in Rats by Active Immunization with Cardiac Myosin
  • Support Protocol 1: Purification of Cardiac Myosin
  • Support Protocol 2: Preparation of Antigen/Adjuvant Emulsion by Agitation for EAM Induction in Mice and Rats
  • Support Protocol 3: Collection and Histopathological Assessment of Mouse or Rat Hearts
  • Support Protocol 4: Immunostaining of Rat Tissues for Deposited Antibody
  • Basic Protocol 3: Induction of Myocarditis in Mice by Inoculation with Coxsackievirus B3 (CVB3) from Infectious cDNA Clones
  • Basic Protocol 4: Induction of Myocarditis and DCM in Mice by Inoculation with Heart‐Passaged Coxsackievirus B3 (CVB3)
  • Basic Protocol 5: Induction of Autoimmune Valvular Heart Disease by Recombinant Streptococcal M Protein
  • Basic Protocol 6: Experimental Prevention of Myocarditis in the Animal Model
  • Human Myocarditis
  • Basic Protocol 7: Protein Kinase A (PKA) Assay of Serum Antibody Signaling of the β Adrenergic Receptor with Subsequent Activation of PKA
  • Basic Protocol 8: Measurement of Serum Antibody Titers Against Human Cardiac Myosin, Human Adrenergic β1 and β2 Receptor, and Muscarinic M2 Receptor
  • Basic Protocol 9: Detection of Antibodies Against Human Cardiac Myosin Peptides in Human Serum
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of EAM in Mice by Active Immunization with Cardiac Myosin

  Materials
  • Cardiac myosin or cardiac myosin peptide (see protocol 4; for peptide sequences; see Table 15.14.3)
  • Complete Freund's adjuvant (CFA; Difco)
  • Pertussis toxin (PTX) stock solution (see recipe), if required, depending on mouse strain (PTX may be used with all strains, but is required for BALB/c EAM)
  • PBS/1% NMS: phosphate‐buffered saline (PBS; appendix 2A), sterile, containing 1% (v/v) normal mouse serum (NMS; unit 1.7; if PTX is being used)
  • Susceptible mice of either sex (See Table 15.14.1), 6 to 8 weeks old
  • Commercially available assay for cTnI or cTnT (optional)
  • 1‐ml glass syringes with Luer‐Lok tips
  • 20‐ and 25‐G bevel‐tipped needles, sterile
  • 1‐ml plastic syringes, with Luer‐Lok tips, sterile
  • Additional reagents and equipment for preparation of cardiac myosin and antigen emulsions (see Support Protocols protocol 41 and protocol 52), mouse restraint (unit 1.3), injection (unit 1.6), blood collection (for measurement of cTnI or cTnT; unit 1.7), euthanasia (unit 1.8), and tissue preparation and histopathology (see protocol 6)

Alternate Protocol 1: Induction of EAM in Mice by Adoptive Transfer of Cardiac Myosin–Stimulated T Cells

  • Donor C.B‐17 mice (6 to 8 weeks old) immunized with cardiac myosin emulsified in CFA (see protocol 1, steps 1 to 6)
  • HBSS+: Hanks' balanced salt solution ( appendix 2A) supplemented with 1% newborn calf serum (NCS), 1% HEPES buffer (e.g., Invitrogen), and 50 µg/ml gentamicin, 4°C
  • Complete RPMI‐10 (see recipe), 37°C
  • Concanavalin A (Con A; type IV, Sigma)
  • Ficoll‐Hypaque (Amersham Pharmacia Biotech)
  • PBS/1% FBS: phosphate‐buffered saline (PBS; appendix 2A) supplemented with 1% (v/v) fetal bovine serum (FBS), sterile filtered
  • SCID recipient mice, 6 to 8 weeks old
  • Disposable tissue culture flasks, sterile
  • 1‐ml plastic Luer‐Lok syringes, sterile
  • 25‐G and 30‐G bevel‐tipped needles, sterile
  • Additional reagents and equipment for harvesting spleens (unit 1.9), preparation of single‐cell suspensions (unit 3.1), cell counting ( appendix 3A), Ficoll/Hypaque gradient centrifugation of spleen cell suspensions (unit 3.1), testing of cell viability by trypan blue exclusion ( appendix 3B), T cell enrichment by nonadherence to nylon (unit 3.2), cytotoxic elimination of B cells and accessory cells (unit 3.3), tail vein injection (unit 1.6), and euthanasia using cervical dislocation or CO 2 (unit 1.8)

Basic Protocol 2: Induction of EAM in Rats by Active Immunization with Cardiac Myosin

  Materials
  • Lewis rats, female, 6 to 8 weeks old
  • Ketamine
  • Xylazine
  • Cardiac myosin or cardiac myosin peptide (immunogen; protocol 4; also see Table 15.14.3)
  • Supplemented CFA: complete Freund's adjuvant supplemented with 10 mg/ml heat‐killed Mycobacterium tuberculosis strain H37Ra (Difco; see unit 15.6 for details of preparation)
  • Bordetella pertussis, heat killed (pertussis cell concentrate; Michigan State Health Department, Lansing, Michigan; optional)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 10% neutral buffered formalin (Fisher Scientific)
  • 1‐ml glass syringes with Luer‐Lok tips
  • 20‐, 23‐, and 25‐G bevel‐tipped needles, sterile
  • 1‐ml plastic syringes, sterile, with Luer‐Lok tips
  • Additional reagents and equipment for anesthesia of rodents (unit 1.4) preparation of cardiac myosin (see protocol 4), preparation of antigen emulsions (see protocol 5), rat restraint (unit 1.3), injections (unit 1.6), euthanasia (unit 1.8), and tissue preparation for histopathology (see protocol 6)

Support Protocol 1: Purification of Cardiac Myosin

  Materials
  • Hearts, freshly harvested or freshly thawed (see protocol introduction above)
  • Buffers A and B (see reciperecipes)
  • Cold distilled water containing DTT and protease inhibitors (leupeptin, PMSF, TLCK, and benzamidine) at the same concentrations as in Buffer A (see recipe for Buffer A)
  • 1 M HCl
  • 3 M KCl stock solution
  • Ammonium sulfate [(NH 4) 2SO 4]
  • 0.1 M ATP stock solution
  • 1 M MgCl 2 stock solution
  • Bradford Protein Assay Solution and standards (BioRad) or equivalent
  • Scissors
  • Polypropylene centrifuge bottles of size appropriate for rotor
  • Tissue homogenizer, 100‐ to 200‐ml capacity
  • High‐speed centrifuge, refrigerated (Sorvall RC‐5B, or equivalent)
  • High‐volume‐capacity rotor (Sorvall GSA, or equivalent)
  • Graduated cylinder of appropriate size
  • Glass wool
  • Stirring plate and magnetic stirrer
  • Ultracentrifuge, refrigerated (Beckman L8‐7OM or equivalent)
  • Ultracentrifuge rotor, 100‐ to 200‐ml capacity, certified for 140,000 × g (e.g., Beckman Ti 45 or equivalent)
  • Additional reagents and equipment for denaturing SDS‐PAGE (unit 8.4)

Support Protocol 2: Preparation of Antigen/Adjuvant Emulsion by Agitation for EAM Induction in Mice and Rats

  Materials
  • Immunogen: cardiac myosin or cardiac myosin peptide (see Table 15.14.3)
  • Complete Freund's adjuvant (CFA, Difco) for mice or supplemented CFA (containing 10 mg/ml heat‐killed M. tuberculosis strain H37Ra, Difco; see unit 15.6) for rats
  • 5‐ to 10‐ml conical‐bottom polypropylene test tubes, sterile
  • Vortex mixer (Vortex Gene‐2, VWR, with microwell platform insert)
  • Glass syringes with Luer‐Lok tips
  • Bevel‐tipped needles: 20‐G, plus 25‐G (for mouse) or 25‐G (for rat)

Support Protocol 3: Collection and Histopathological Assessment of Mouse or Rat Hearts

  Materials
  • Mouse (see protocol 1 or protocol 2) or rat (see protocol 3) with EAM
  • 70% ethanol
  • 10% formalin, neutral buffered
  • Dissecting scissors (blunt and sharp) and forceps
  • Single‐edged razor blades
  • Tissue cassettes: Monosette IV (VWR) or equivalent for mice or Mega‐Cassette (VWR) or equivalent for rats
  • No. 2 pencil
  • Additional reagents and equipment for euthanasia (unit 1.8), paraffin embedding and sectioning (unit 21.4), and hematoxylin and eosin staining (unit 12.8)

Support Protocol 4: Immunostaining of Rat Tissues for Deposited Antibody

  Materials
  • Formalin‐fixed, paraffin‐embedded heart tissue sections (see unit 20.6)
  • Mouse anti–rat IgG, IgG1, IgG2a and isotype control mouse IgG antibodies (Sigma‐Aldrich)
  • Biotin‐conjugated goat anti–mouse IgG antibody or rabbit anti‐goat IgG (Jackson Immunoresearch Laboratories)
  • Alkaline phosphatase–conjugated streptavidin (Jackson Immunoresearch Laboratories)
  • Fast Red substrate (BioGenex)
  • Mayer's hematoxylin (BioGenex)
  • H9c2 rat heart cell‐line (ATCC #CRL‐1446)
  • Serum from cardiac myosin–immunized Lewis rat ( protocol 3)
  • 10% neutral buffered formalin (Fisher Scientific)
  • Xylene or CitriSolv (Fisher Scientific, cat. no. 22‐143‐975)
  • Ethanol
  • 0.1 M citrate buffer, pH 6 (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2 mg/ml crystal violet in ethanol
  • Apo Active 3 kit (Cell Technology, cat. no. FAB200‐2; http://celltechnology.com/)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.1% (v/v) Triton X‐100
  • Rabbit anti–active caspase 3 antibody (supplied with Apo Active 3 kit)
  • 2% (w/v) bovine serum albumin (BSA) in PBS (see appendix 2A for PBS)
  • Goat anti‐rabbit FITC‐labeled antibody (supplied with Apo Active 3 kit))
  • 4‐well cell culture chamber slides
  • Conventional microwave oven
  • Fluorescence microscope
  • Additional reagents and equipment for deparaffinizing tissue (unit 21.6)

Basic Protocol 3: Induction of Myocarditis in Mice by Inoculation with Coxsackievirus B3 (CVB3) from Infectious cDNA Clones

  Materials
  • Infectious cDNA plasmid (see Table 15.14.5)
  • PureYield Plasmid Midiprep System (Promega)
  • HeLa cells (ATCC #CCL‐2)
  • Lipofectamine LTX with PLUS Reagent (Invitrogen/Life Technologies)
  • Opti‐MEM I Reduced Serum Medium (Invitrogen/Life Technologies, cat. no. 31985088)
  • Complete MEM‐5 medium (see recipe)
  • Ethanol/dry ice slurry (optional)
  • Complete RPMI‐2 (with 2% FBS) and RPMI‐5 (with 5% FBS) (see recipe)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 1× trypsin‐EDTA (0.05% trypsin/0.53 µM EDTA; Invitrogen/Life Technologies, cat. no. 25300‐062)
  • 3% agar (made by autoclaving 3 g agar in 100 m distilled H 2O)
  • 10% neutral buffered formalin (Fisher Scientific)
  • 2% (w/v) crystal violet in 20% ethanol
  • Mice (see text over step 24, below)
  • 70% ethanol
  • 75‐ and 150 cm2 tissue culture flasks
  • Cell scrapers
  • 50‐ml conical tubes (e.g., BD Falcon)
  • 0.5‐ml microcentrifuge tubes
  • 60 × 15–mm plastic petri dishes (sterile)
  • 96‐well round‐bottom tissue culture plate
  • 100°C water bath
  • 10‐ml syringes and 26‐G needles
  • Blunt dissection scissors
  • Forceps
  • Suture for ligation
  • Gauze pads
  • Balance for weighing the heart
  • Razor blade
  • Tissue cassettes
  • Tissue homogenizer (pestle motor, VWR cat. no. 47747‐370; pestle, VWR cat. no. 47747‐366)
  • 12 × 75–mm tubes (optional)
  • Centrifuge
  • Additional reagents and equipment for injection of mice (unit 1.6), euthanasia of mice (unit 1.8), immunohistochemistry (unit 21.4), and hematoxylin/eosin staining (unit 12.8)
    Table 5.4.5   MaterialsInfectious cDNA Clones of Coxsackievirus B

    Clone/plasmid Characteristic Reference
    CVB3(Nancy) Myocarditic Kandolf and Hofschneider ( )
    CVB3m Myocarditic Chapman et al. ( ); Lee et al. ( )
    CVB3/0 Amyocarditic
    CVB3(pH3) Myocarditic Knowlton et al. ( )
    CVB3(pH310A1) Amyocarditic
    CVB3 Myocarditic Cameron‐Wilson et al. ( )
    pMKS1 pH3 with added sfi 1 site Slifka et al. ( )
    pMKS2 Db‐restricted LCMV GP 33‐41
    pMKS3 Lb‐restricted LCMV NP 118‐126
    pCVB‐GPTh IAb‐restricted LCMV GP 61‐80 Kemball et al. ( ); Tsueng et al. ( )
    eGFP‐CVB3 pMKS1 with inserted GFP Feuer et al. ( )
    dsRed‐CVB3 pMKS1 with inserted Ds‐Red Cornell et al. ( )

Basic Protocol 4: Induction of Myocarditis and DCM in Mice by Inoculation with Heart‐Passaged Coxsackievirus B3 (CVB3)

  Materials
  • Vero cells (ATCC #CCL‐81)
  • Complete MEM‐10 (see recipe)
  • Complete MEM‐2 (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • CVB3, Nancy strain (ATCC #VR‐30) or tissue culture–derived virus (see protocol 9)
  • Bleach
  • Mice aged 6 to 10 weeks old
  • Methyl cellulose in MEM‐2 (see recipe)
  • 1% (w/v) methylene blue in 10% phosphate‐buffered formalin
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 10% phosphate‐buffered formalin (Decal Corp., cat. no. 2101, http://www.decal‐bone.com/)
  • Masson's trichrome (optional)
  • Centrifuge
  • Additional reagents and equipment for injection of mice (unit 1.6), immunohistochemistry (unit 21.4), and hematoxylin and eosin staining (unit 12.8)

Basic Protocol 5: Induction of Autoimmune Valvular Heart Disease by Recombinant Streptococcal M Protein

  Materials
  • Lewis rats, 8 weeks old
  • Purified recombinant group A streptococcal M protein (Fischetti et al., )
  • Supplemented CFA: complete Freund's adjuvant supplemented with 5 mg/ml heat‐killed Mycobacterium tuberculosis strain H37Ra (Difco; see unit 15.6 for details of preparation)
  • Bordetella pertussis
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Incomplete Freund's adjuvant (IFA; Sigma)
  • Additional reagents rat restraint (unit 1.3), injections (unit 1.6), preparation of antigen emulsions ( protocol 5), euthanasia (unit 1.8), and tissue preparation for histopathology (see protocol 6)

Basic Protocol 6: Experimental Prevention of Myocarditis in the Animal Model

  Materials
  • H9c2 cells (ATCC #CRL‐1446)
  • Complete IMDM‐10 (see recipe)
  • Sample of interest (serum from human cardiac patients)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Serum‐free IMDM medium (e.g., Invitrogen/Life Technologies)
  • Protein extraction buffer (see recipe)
  • 10 µCi/µl [γ‐32P]ATP (3000 Ci/mmol)
  • 2 M NaCl
  • 2 M NaCl in 1% H 3PO 4
  • Bradford Protein Assay Solution and standards (Bio‐Rad or equivalent)
  • 75‐cm2 (T‐75) tissue culture flasks
  • Cell scraper
  • 14‐ml culture tubes (e.g., VMR, cat. no. 60818‐703)
  • Homogenizer (chilled): Dounce homogenizer or similar homogenizer for cultured cells
  • 30°C heating block or water bath
  • Washing container (plastic utility box, 19 × 15 × 10 cm)
  • Scintillation counter or phosphor imaging device
  • Orbital platform shaker(optional)
  • Heat lamp (optional)
CAUTION: The [γ32P] ATP must be handled as per safety regulations for the use of radioactivity. You must work behind a safety shield and minimize exposure at all times. Gloves and safety goggles should be worn at all times. Also see appendix 1Q.

Basic Protocol 7: Protein Kinase A (PKA) Assay of Serum Antibody Signaling of the β Adrenergic Receptor with Subsequent Activation of PKA

  Materials
  • Antigens:
    • Human Adrenergic Beta 1 Receptor (Perkin Elmer)
    • Human Adrenergic Beta 2 Receptor (Perkin Elmer)
    • Human Muscarinic M 2 Receptor (Perkin Elmer)
    • Human Cardiac Myosin
  • Carbonate‐bicarbonate coating buffer (see recipe)
  • PBS/0.05% Tween: Phosphate‐buffered saline (PBS; appendix 2A) containing 0.05% (v/v) Tween 20 plus 100 µg/ml sodium azide
  • 1% BSA/PBS: phosphate‐buffered saline (PBS; appendix 2A) containing 1% (w/v) bovine serum albumin (BSA) plus 100 µg/ml sodium azide
  • Sample of interest (serum from human cardiac patients)
  • Secondary antibody: affinity‐purified, Fc‐specific, alkaline phosphatase–conjugated goat anti–human IgG: Sigma, cat. no. A3312 should be used for the β 1, β 2, and M 2 receptors; Jackson Immunoresearch, cat. no. 109‐056‐097 should be used for human cardiac myosin
  • Substrate solution: 1 mg/ml p‐nitrophenyl phosphate (Sigma, cat. no. S0942) in diethanolamine developing buffer (see recipe)
  • Multichannel pipettor and disposable tips
  • 96‐well ELISA plates (Immulon IV flat bottom plates, Thermo Scientific)
  • Plastic wrap
  • ELISA plate washer or plastic squirt bottles
  • Refrigerated centrifuge with microtiter plate carrier
  • Microtiter plate reader, spectrophotometer with 405 filter

Basic Protocol 8: Measurement of Serum Antibody Titers Against Human Cardiac Myosin, Human Adrenergic β1 and β2 Receptor, and Muscarinic M2 Receptor

  Materials
  • Human cardiac myosin peptides (see list in protocol introduction, above)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 100 µg/ml sodium azide
  • Carbonate/bicarbonate coating buffer (see recipe)
  • PBS/0.05% Tween: phosphate‐buffered saline (PBS; appendix 2A) containing 0.05% (v/v) Tween 20 plus 100 µg/ml sodium azide
  • 1% BSA/PBS: phosphate‐buffered saline (PBS; appendix 2A) containing 1% (w/v) bovine serum albumin (BSA)
  • Sample of interest (serum from human cardiac patients)
  • Secondary antibody: affinity‐purified, Fc‐specific, alkaline phosphatase–conjugated goat anti–human IgG (Sigma, cat. no. A3312)
  • Substrate solution: 1 mg/ml p‐nitrophenyl phosphate (Sigma, cat. no. S0942) in diethanolamine developing buffer (see recipe)
  • Multichannel pipet and disposable tips
  • 96‐well ELISA plates (Immulon IV flat‐bottom plates, Thermo Scientific)
  • Plastic wrap
  • ELISA washer or plastic squirt bottles
  • Refrigerated centrifuge with microtiter plate carrier
  • Microtiter plate reader spectrophotometer with 405‐nm filter
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Figures

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Literature Cited

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