Complete congenital heart block is associated with increased autoantibody titers against calreticulin

Orth, Thomas; Dörner, Thomas; Büschenfelde, K H Meyer zum; Mayet, W.‐J.

doi:10.1046/j.1365-2362.1996.120270.x

Cited by 44 publications

(23 citation statements)

References 27 publications

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“…A previous study has suggested that complete heart block may be associated with production of an autoantibody against calreticulin (16,17). Therefore, we investigated the possibility that overexpression of calreticulin in the transgenic mice might lead to secretion of the protein and/or production of anti-calreticulin antibod-…”

Section: Resultsmentioning

confidence: 99%

Complete heart block and sudden death in mice overexpressing calreticulin

Nakamura¹,

Robertson²,

Liu³

et al. 2001

J. Clin. Invest.

125

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The expression of calreticulin, a Ca 2+ -binding chaperone of the endoplasmic reticulum, is elevated in the embryonic heart, and because of impaired cardiac development, knockout of the Calreticulin gene is lethal during embryogenesis. The elevated expression is downregulated after birth. Here we have investigated the physiological consequences of continued high expression of calreticulin in the postnatal heart, by producing transgenic mice that overexpress the protein in the heart. These transgenic animals exhibit decreased systolic function and inward I Ca,L , low levels of connexin43 and connexin40, sinus bradycardia, and prolonged atrioventricular (AV) node conduction followed by complete heart block and sudden death. We conclude that postnatal downregulation of calreticulin is essential in the development of the cardiac conductive system, in particular in the sinus and AV nodes, when an inward Ca 2+ current is required for activation. This work identifies a novel pathway of events, leading to complete heart block and sudden cardiac death, which involves high expression of calreticulin in the heart. G AC TAC AG C T C G T C C T T G G C C T G T C TAG -CATAGTCAGGAACATCATATGGGTAC-3′were annealed to create double strand DNA fragmentepitope is underlined followed by KDEL ER retrieval signal). cDNA encoding calreticulin was synthesized by a PCR-driven amplification (8) and cloned into SalI site of a plasmid containing the 5.5-kb mouse cardiac α-myosin heavy chain (α-MHC) (Figure 1a). Linearized pBS-α-MHC-CRT-HA was microinjected into the fertilized oocytes, which were transferred into the oviduct of pseudopregnant FVB/N mice. Transgenic mice were identified by PCR analysis of tail genomic DNA using a forward primer corresponding the 5′ end of the mouse the α-MHC promoter sequence (MHCf: 5′-TATCTCCCCCATAAGAGTTT-3′) and a reverse primer corresponding to the 5′ end of the calreticulin cDNA sequence (CRT-N3A: 5′-GTCAATCTTCACCTCAT-ACG-3′) (Figure 1a). Founder mice were identified, bred with wild-type FVB/N mice, and maintained in a pathogen-free environment.SDS-PAGE and Western immunoblotting. Proteins from mouse tissues including heart, brain, lung, liver, kidney, and thymus were lysed, separated by SDS-PAGE followed by immunoblotting (9). Protein assays were carried out using DC Protein Assay kit (Bio-Rad Laboratories Inc., Hercules, California, USA). Blots were probed with rabbit anti-HA antibodies, goat or rabbit anti-calreticulin antibodies (9, 10), rabbit anti-calnexin (Stress Gene, Victoria, British Columbia, Canada; 1:500 dilution), rabbit anti-BiP (1:2,000 dilution), rabbit anti-PDI (9) (1:500 dilution), rabbit anti-calsequestrin (10) (1:300 dilution), rabbit anti-Cx43 (1:20,000 dilution), or rabbit anti-SERCA2 antibodies (11) (1:1,000 dilution). Antibody binding was detected with appropriate peroxidase-conjugated secondary antibodies followed by a standard enhanced chemiluminescence development reaction.Northern blot analysis. Northern blot analysis was carried out as described elsewhere (10). The following c...

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Section: Resultsmentioning

confidence: 99%

Complete heart block and sudden death in mice overexpressing calreticulin

Nakamura¹,

Robertson²,

Liu³

et al. 2001

J. Clin. Invest.

125

View full text Add to dashboard Cite

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“…The autoantibody-mediated immune response to calreticulin isoforms was found in pancreatic [24] and gastric cancer [25]. Increased levels of IgA and/or IgG anti-calreticulin antibodies in the sera of many patients with HCC and colorectal adenocarcinoma and 47% of patients with pancreatic adenocarcinoma (PACA) were found.…”

Section: Introductionmentioning

confidence: 99%

Overexpression of Calreticulin in Malignant and Benign Breast Tumors: Relationship with Humoral Immunity

et al. 2012

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Objective: Calreticulin is a multicompartmental protein which regulates many important cellular responses. The aim of this study was to elucidate whether the intensity and location of calreticulin overexpression in tumor cells are related to the elevated humoral immunity to calreticulin in patients with benign or malignant breast disease. Methods: This study involved 27 patients with benign and 58 patients with malignant breast tumors before surgical resection and 38 healthy volunteers. Cytoplasmatic or membranous calreticulin overexpression in malignant or benign cells in paraffin-embedded tissues was determined using immunohistochemistry. Levels of the serum anti-calreticulin autoantibodies were detected by ELISA. Results: Statistically significant differences between serum levels of IgA of anti-calreticulin antibodies in controls and patients with breast tumors, and between controls and patients with nonmalignant breast diseases were found, but no statistically significant differences were found between levels of serum IgG anti-calreticulin antibodies. Humoral immunity to calreticulin developed against cytoplasmatic and co-localized membranous calreticulin was not correlated to the intensity of its overexpression and was present even in the absence of its membranous localization. Conclusions: The degree of calreticulin overexpression in lobular breast carcinoma is lower than in ductal breast carcinoma. Elevated concentrations of anti-calreticulin IgA antibodies were present more frequently in patients with metastasis in locoregional lymph nodes in comparison to anti-calreticulin IgG antibodies.

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“…[1][2][3] In the ER, CRT has a number of functions, including Ca 2+ sensing, protein folding and a role in MHC class I assembly. 4 The multifunctional nature of CRT suggests that it may be prevalent in many cellular compartments.…”

Section: Introductionmentioning

confidence: 99%

A Mechanism of Release of Calreticulin from Cells During Apoptosis

Tarr

Young

Morse

et al. 2010

Journal of Molecular Biology

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Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca 2+ homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca 2+ -dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-L-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.

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Complete congenital heart block is associated with increased autoantibody titers against calreticulin

Cited by 44 publications

References 27 publications

Complete heart block and sudden death in mice overexpressing calreticulin

Complete heart block and sudden death in mice overexpressing calreticulin

Overexpression of Calreticulin in Malignant and Benign Breast Tumors: Relationship with Humoral Immunity

A Mechanism of Release of Calreticulin from Cells During Apoptosis

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