Abstract

Dystrophic calcification (DC), or extra-osseous deposition of calcium phosphate,

is associated with numerous cardiovascular diseases ranging from acute

conditions such as myocardial infarction to chronic conditions including

atherosclerosis. Development of DC in mouse models is multifactorial, with diet,

age and genotype each contributing to the disease phenotype. Although DC has

been characterized in several mouse strains, the relationship between

inflammation and DC has not been addressed and no autoimmune component

has been identified. We identified a substrain of BALB/c mice (BALB/cByJ) that

spontaneously develop epicardial DC lesions containing focal deposits of

calcium and infiltrating mononuclear cells. Bone marrow chimera experiments

using GFP-expressing donor bone marrow revealed that the lesions are strongly

GFP positive, suggesting an inflammatory component of the disease. In

particular, there was substantial GFP positive cell infiltration at the site of

calcinosis. A characterization of the infiltrating cells by immunohistochemistry

confirmed the presence of immune cells. Initiation of experimental autoimmune

myocarditis in these animals led to the formation of a more pronounced lesion,

where the calcinosis appeared to serve as a focus of the myocarditis. Further

study of this model may help elucidate the dynamic interplay between

inflammation and calcification in cardiovascular disease.

Conclusions

BALB/cByJ mice develop spontaneous cardiac lesions at

a young age, high prevalence and high severity.

Bone marrow derived cells, including: CD3, CD4, and CD11b positive

cells, are present within lesions.

The disease does not appear to correlate with a known

mutation associated with calcinosis in mice.

Induction of autoimmune myocarditis in animals enhances surface

lesion severity, suggesting a link to inflammation.

Aaron M. Glass, Wanda Coombs, Nedal Darwish and Steven M. Taffet

Figure 1. BALB/cByJ mice develop spontaneous cardiac surface lesions

Hearts from 5 week old animals exhibit pronounced lesions, primarily in the layers of myocardium immediately deep

to the epicardium of the right ventricular wall. A and B. Dissecting microscope photomicrographs taken from a non-

afflicted (A) and a moderately-afflicted animal (B). C. Comparison of lesional area as a percentage of total

ventricular surface area for male BALB/c (Taconic) and BALB/cByJ (Jackson) hearts at 3, 5 and 12 weeks. Images

similar to those shown in panels “A” and “B” were measured for total surface area and lesional area using ImageJ

(NIH) software. Each symbol represents data averaged from 4 aspects of a single heart. Analyzed by ANOVA

followed by Bonferroni’s post-test, *p<.05, **p<.01. D. Alizarin Red S staining of 5 week old BALB/cByJ heart.

Red/brown areas indicate that the lesions include calcium deposits. E. Masson’s trichrome staining of heart shown

in panel “D”. Blue/purple staining indicates collagen is present in the lesion. BALB/cByJ mice exhibit

spontaneous cardiac lesions that are visible on the ventricular surface and occupy a significant proportion

of the total ventricular surface area by 5 weeks of age. Generally limited to the right ventricular epicardial

surface, these lesions are characterized by the presence of calcium deposits and marked fibrosis.

AcknowledgementsWe wish to sincerely thank Dr. Michael Lyon and Linda Steer for their expertise and the use of their cryostat; the Sandy Hayes lab for their generous gifts of antibodies and advice; Dr. Mira Krendel for the use of her dissecting microscope;

and Li Gao and Chris Burrer for their technical expertise and hard work – without these contributions, this work would not have been possible. This work was supported by research grants # HL039707 and #HL100111 from the NIH.

Role of bone marrow-derived cells and inflammation in

spontaneous cardiac dystrophic calcification

EF

Bagg, ~1913

Snell, 1932

Strong

Little, 1909

ScottAndervont,

1935

Taconic, 1997

NIH, 1951

Jackson,

1947

Bailey, ~1960

/cByJ /cAnNCR /cTac /cJ

C3H DBA C57BL

Little, ~1921

BALB

/HeJ /2J /6

A. Develops Dystrophic Cardiac Calcinosis:

+++ - + + +1 ++1 -1

B. Carries DCC related Abcc6 mutation:

- NT - NT + + -

C. Qa-2 status:

-/- -/- -/- +/- NT NT +/+

CD3 CD3

CD3CD11b

CD3CD4

A B C

D E

Figure 4. BALB/cByJ dystrophic calcification is not related to Abcc6 mutation or Qa-2 status

A genetic etiology has been suggested to play a major role in the development of cardiac calcinosis in mice.

Top. A basic pedigree of the mouse strains examined in this study. Names indicate investigators or organizations

credited with maintaining various strains beginning at the dates shown. Bottom. A. Prevalence of lesions among

male animals in listed strains, “-” = none detected, “+” = 1 to 30% of animals studied, “++” = 31 to 60%, “+++” = 60

to 100%. B. Genomic DNA from animals of each strain was isolated and a region of exon 14 of the Abcc6 gene was

amplified and sequenced. This region is known to contain a polymorphism that has been linked to calcinosis in C3H

and DBA mice2. “+” = strain carries the calcinosis allele, “-” = calcinosis allele absent, “NT” = strain not tested.

C. Several BALB/c substrains are known to lack expression of the minor histocompatability molecule Qa-2, which

has been suggested to have an immunoregulatory function. Qa-2 status was analyzed by flow cytometry using

mouse anti-Qa-2 antibody (eBioscience). “-/-” = no detectable expression, “+/-” = mid-level expression, “+/+” = high

expression, “NT” = not tested. Cardiac calcinosis in BALB/cByJ mice appears to have an etiology distinct

from that of related strains, such as DBA and C3H. Development of disease is not related to the absence of

the immunoregulatory minor histocompatibility antigen Qa-2.

1. Eaton GJ et al. Dystrophic cardiac calcinosis in mice: genetic, hormonal, and dietary influences. American Journal of Pathology. 1978.

2. Aherrahrou Z et al. An alternative splice variant in Abcc6, the gene causing dystrophic calcification, leads to protein deficiency in C3H/He mice. Journal

of Biological Chemistry. 2008.

Figure 5. Induction of experimental autoimmune

myocarditis (EAM) enhances lesion severity

It has been suggested that inflammation or infection may

exacerbate, or even initiate, the development of cardiac

calcinosis in predisposed mouse strains. Animals were treated

with dendritic cells loaded with a peptide fragment of α myosin

heavy chain (BALB/c and /cByJ Treated) or a fragment of

ovalbumin (/cByJ Control), followed by a booster of respective

peptide with an adjuvant. Animals were sacrificed and hearts

were excised 21 days after the initiation of treatment. Hearts

were imaged and analyzed as in “Figure 1”. *p<.05. EAM-

induced animals develop significantly more severe

lesions than control animals or untreated animals (as

shown in Figure 1C).

EAM protocol based on: Blyszczuk et al. Myeloid differentiation factor-

88/interleukin-1 signaling controls cardiac fibrosis and heart failure progression in

inflammatory dilated cardiomyopathy. Circulation Research. 2009.

Figure 3. Characterization of immune cells present in lesions by immunofluorescence microscopy

Frozen sections were prepared from the hearts from 5 week old animals. Sections were probed with rabbit anti-CD3

antibody (Abcam) to identify T lymphocytes, rat anti-CD11b (Abcam) to identify granulocytes/macrophages, and rat

anti-CD4 (BioLegend) to identify TH cells. The sections were subsequently probed with goat anti-rabbit Alexa Fluor

488 (Invitrogen) and/or goat anti-rat Alexa Fluor 594 for fluorescent immunodetection. Staining was also performed

for CD8 positive cells, but were rarely observed in these lesions (not shown). Images were produced on a Nikon

Eclipse E800 fluorescence microscope equipped with a Spot RT Slider (Diagnostic Instruments) digital camera.

Lesions contain focal areas of CD3, CD4 and CD11b positive cells, suggesting the presence of T cells and

macrophages/granulocytes. Many CD4+ cells were present, but CD8+ cells were rarely observed.

BALB substrain*Lesion

prevalence†

% of hearts

with lesions

/cJ (Jackson) 1/6 16.7

/cAnNCR (NIH) 0/7 0

/c (Taconic) 1/17 5.8

/cByJ (Jackson) 28/33 84.8Table 1. Prevalence of epicardial lesions in selected BALB/c mouse substrains at 5 – 6 weeks of age

Male animals were euthanized and their hearts were examined by dissecting microscopy for any evidence of

epicardial lesions. *Animal supplier is indicated in parentheses. †Prevalence indicates number of afflicted animals

with detectable lesions/total number of animals examined. At 5 weeks of age, BALB/cByJ animals exhibit a

dramatically higher incidence of epicardial lesions than several related BALB/c substrains.

White Light GFP

5x106 BM

cellsGFP +

(donor) 10 wksc/ByJ

(recipient)chimera

Figure 2. Cardiac lesions of BALB/cByJ-GFP

chimeric mice exhibit strong GFP fluorescence.

GFP chimeras were generated using donor bone

marrow cells from GFP+ BALB/cByJ mice

(Jackson). Cells were transferred to irradiated (900

Rad), 3 week old BALB/cByJ recipients after which

the animals were allowed 10 weeks for

reconstitution. After reconstitution, animals were

euthanized and hearts were excised and imaged

for GFP surface fluorescence using an IVIS 50

imaging system (Caliper/Xenogen). Flow

cytometric analysis of peripheral blood showed

that an average of 78.4% ± 2.3% of the CD45+

cells were also GFP+, indicating successful

reconstitution. These results indicate that the

cardiac lesions contain high concentrations of

infiltrating, bone marrow derived cells, possibly

implicating an autoimmune mechanism.