Calreticulin Deficiency Disturbs Ribosome Biogenesis and Results in Retardation in Embryonic Kidney Development.

Nephrogenesis is driven by complex signaling pathways that control cell growth and differentiation. The endoplasmic reticulum chaperone calreticulin (Calr) is well known for its function in calcium storage and in the folding of glycoproteins. Its role in kidney development is still not understood. We provide evidence for a pivotal role of Calr in nephrogenesis in this investigation. We show that Calr deficiency results in the disrupted formation of an intact nephrogenic zone and in retardation of nephrogenesis, as evidenced by the disturbance in the formation of comma-shaped and s-shaped bodies. Using proteomics and transcriptomics approaches, we demonstrated that in addition to an alteration in Wnt-signaling key proteins, embryonic kidneys from Calr-/- showed an overall impairment in expression of ribosomal proteins which reveals disturbances in protein synthesis and nephrogenesis. CRISPR/cas9 mediated knockout confirmed that Calr deficiency is associated with a deficiency of several ribosomal proteins and key proteins in ribosome biogenesis. Our data highlights a direct link between Calr expression and the ribosome biogenesis.

Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin.

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.

Calreticulin haploinsufficiency augments stem cell activity and is required for onset of myeloproliferative neoplasms in mice.

Mutations in JAK2, myeloproliferative leukemia virus (MPL), or calreticulin (CALR) occur in hematopoietic stem cells (HSCs) and are detected in more than 80% of patients with myeloproliferative neoplasms (MPNs). They are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calr haploinsufficiency promoted the self-renewal capacity of HSCs. We generated CALRdel52 mutant transgenic mice with Calr haploinsufficiency as a model that mimics human MPN patients and found that Calr haploinsufficiency restored the self-renewal capacity of HSCs damaged by CALR mutations. Only recipient mice transplanted with Lineage-Sca1+c-kit+ cells harboring both CALR mutation and Calr haploinsufficiency developed MPN in competitive conditions, showing that CALR haploinsufficiency was necessary for the onset of CALR-mutated MPNs.

Loss of Calreticulin Uncovers a Critical Role for Calcium in Regulating Cellular Lipid Homeostasis.

A direct link between Ca2+ and lipid homeostasis has not been definitively demonstrated. In this study, we show that manipulation of ER Ca2+ causes the re-distribution of a portion of the intracellular unesterified cholesterol to a pool that is not available to the SCAP-SREBP complex. The SREBP processing pathway in ER Ca2+ depleted cells remained fully functional and responsive to changes in cellular cholesterol status but differed unexpectedly in basal activity. These findings establish the role of Ca2+ in determining the reference set-point for controlling cellular lipid homeostasis. We propose that ER Ca2+ status is an important determinant of the basal sensitivity of the sterol sensing mechanism inherent to the SREBP processing pathway.

Calreticulin secures calcium-dependent nuclear pore competency required for cardiogenesis.

Calreticulin deficiency causes myocardial developmental defects that culminate in an embryonic lethal phenotype. Recent studies have linked loss of this calcium binding chaperone to failure in myofibrillogenesis through an as yet undefined mechanism. The purpose of the present study was to identify cellular processes corrupted by calreticulin deficiency that precipitate dysregulation of cardiac myofibrillogenesis related to acquisition of cardiac phenotype. In an embryonic stem cell knockout model, calreticulin deficit (crt(-/-)) compromised nucleocytoplasmic transport of nuclear localization signal-dependent and independent pathways, disrupting nuclear import of the cardiac transcription factor MEF2C. The expression of nucleoporins and associated nuclear transport proteins in derived crt(-/-) cardiomyocytes revealed an abnormal nuclear pore complex (NPC) configuration. Altered protein content in crt(-/-) cells resulted in remodeled NPC architecture that caused decreased pore diameter and diminished probability of central channel occupancy versus wild type counterparts. Ionophore treatment of impaired calcium handling in crt(-/-) cells corrected nuclear pore microarchitecture and rescued nuclear import resulting in normalized myofibrillogenesis. Thus, calreticulin deficiency alters nuclear pore function and structure, impeding myofibrillogenesis in nascent cardiomyocytes through a calcium dependent mechanism. This essential role of calreticulin in nucleocytoplasmic communication competency ties its regulatory action with proficiency of cardiac myofibrillogenesis essential for proper cardiac development.

Disrupted WNT signaling in mouse embryonic stem cells in the absence of calreticulin.

The role of endoplasmic reticulum (ER) homeostasis and protein quality control in the regulation of WNT signaling is not understood. Here we provide evidence for a role of calreticulin in the regulation of WNT signaling. We show that a deficiency in calreticulin disrupted WNT signaling, and prevented cell cycle progression via the miR-302 microRNA family. These effects were dependent on the Ca(2+) buffering capacity of calreticulin, as the protein is important in regulating ER Ca(2+) release and activation of Ca(2+)-dependent kinase and phosphatase cascades (including c-Src, Akt, and PTP1B). We also show that calreticulin plays a role in the secretion and ER retention of WNT3a, thereby affecting downstream WNT signaling. In calreticulin-deficient ES cells, the WNT and miR-302 dependent maintenance of the naïve ES cell state and the transition to primed pluripotency transition were lost, preventing cells from undergoing accurate differentiation. Together, these findings demonstrate unexpected roles of calreticulin and ER Ca(2+) homeostasis/signaling in the canonical WNT signaling pathway.

Specialization of endoplasmic reticulum chaperones for the folding and function of myelin glycoproteins P0 and PMP22.

Peripheral myelin protein 22 (PMP22) and protein 0 (P0) are major peripheral myelin glycoproteins, and mutations in these two proteins are associated with hereditary demyelinating peripheral neuropathies. Calnexin, calreticulin, and ERp57 are critical components of protein quality control responsible for proper folding of newly synthesized glycoproteins. Here, using confocal microscopy, we show that cell surface targeting of P0 and PMP22 is not affected in the absence of the endoplasmic reticulum chaperones. However, the folding and function (adhesiveness) of PMP22 and P0, measured using the adhesion assay, are affected significantly in the absence of calnexin but not in the absence of calreticulin. Deficiency in oxidoreductase ERp57 results in impaired folding and function of P0, a disulfide bond-containing protein, but does not have any effect on folding or function of PMP22 (a protein that does not contain a disulfide bond). We concluded that calnexin and ERp57, but not calreticulin, play an important role in the biology of peripheral myelin proteins PMP22 and P0, and, consequently, these chaperones may contribute to the pathogenesis of peripheral neuropathies and the diversity of these neurological disorders.

Endoplasmic reticulum calcium depletion impacts chaperone secretion, innate immunity, and phagocytic uptake of cells.

A number of immunological functions are ascribed to cell surface-expressed forms of the endoplasmic reticulum (ER) chaperone calreticulin (CRT). In this study, we examined the impact of ER stress-inducing drugs upon cell surface CRT induction and the resulting immunological consequences. We showed that cell surface expression of CRT and secretion of CRT, BiP, gp96, and PDI were induced by thapsigargin (THP) treatment, which depletes ER calcium, but not by tunicamycin treatment, which inhibits protein glycosylation. Surface expression of CRT in viable, THP-treated fibroblasts correlated with their enhanced phagocytic uptake by bone marrow-derived dendritic cells. Incubation of bone marrow-derived dendritic cells with THP-treated fibroblasts enhanced sterile IL-6 production and LPS-induced generation of IL-1ß, IL-12, IL-23, and TNF-α. However, extracellular CRT is not required for enhanced proinflammatory responses. Furthermore, the pattern of proinflammatory cytokine induction by THP-treated cells and cell supernatants resembled that induced by THP itself and indicated that other ER chaperones present in supernatants of THP-treated cells also do not contribute to induction of the innate immune response. Thus, secretion of various ER chaperones, including CRT, is induced by ER calcium depletion. CRT, previously suggested as an eat-me signal in dead and dying cellular contexts, can also promote phagocytic uptake of cells subject to ER calcium depletion. Finally, there is a strong synergy between calcium depletion in the ER and sterile IL-6, as well as LPS-dependent IL-1ß, IL-12, IL-23, and TNF-α innate responses, findings that have implications for understanding inflammatory diseases that originate in the ER.

Calreticulin is crucial for calcium homeostasis mediated adaptation and survival of thick ascending limb of Henle's loop cells under osmotic stress.

The thick ascending limb of Henle's loop (TALH) is normally exposed to variable and often very high osmotic stress and involves different mechanisms to counteract this stress. ER resident calcium binding proteins especially calreticulin (CALR) play an important role in different stress balance mechanisms. To investigate the role of CALR in renal epithelial cells adaptation and survival under osmotic stress, two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry and functional proteomics were performed. CALR expression was significantly altered in TALH cells exposed to osmotic stress, whereas renal inner medullary collecting duct cells and interstitial cells exposed to hyperosmotic stress showed no significant changes in CALR expression. Moreover, a time dependent downregulation of CALR was accompanied with continuous change in the level of free intracellular calcium. Inhibition of the calcium release, through IP3R antagonist, prevented CALR expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. Overexpression of wild type CALR in TALH cells resulted in significant decrease in cell viability under hyperosmotic stress. In contrast, the hyperosmotic stress did not have any effect on cells overexpressing the CALR mutant, lacking the calcium-binding domain. Silencing CALR with siRNA significantly improved the cell survival under osmotic stress conditions. Taken together, our data clearly highlight the crucial role of CALR and its calcium-binding role in TALH adaptation and survival under osmotic stress.

7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin.

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ~20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.

Decoded calreticulin-deficient embryonic stem cell transcriptome resolves latent cardiophenotype.

Genomic perturbations that challenge normal signaling at the pluripotent stage may trigger unforeseen ontogenic aberrancies. Anticipatory systems biology identification of transcriptome landscapes that underlie latent phenotypes would offer molecular diagnosis before the onset of symptoms. The purpose of this study was to assess the impact of calreticulin-deficient embryonic stem cell transcriptomes on molecular functions and physiological systems. Bioinformatic surveillance of calreticulin-null stem cells, a monogenic insult model, diagnosed a disruption in transcriptome dynamics, which re-prioritized essential cellular functions. Calreticulin-calibrated signaling axes were uncovered, and network-wide cartography of undifferentiated stem cell transcripts suggested cardiac manifestations. Calreticulin-deficient stem cell-derived cardiac cells verified disorganized sarcomerogenesis, mitochondrial paucity, and cytoarchitectural aberrations to validate calreticulin-dependent network forecasts. Furthermore, magnetic resonance imaging and histopathology detected a ventricular septal defect, revealing organogenic manifestation of calreticulin deletion. Thus, bioinformatic deciphering of a primordial calreticulin-deficient transcriptome decoded at the pluripotent stem cell stage a reconfigured multifunctional molecular registry to anticipate predifferentiation susceptibility toward abnormal cardiophenotype.

Evidence for calreticulin attenuation of cardiac hypertrophy induced by pressure overload and soluble agonists.

While calreticulin has been shown to be critical for cardiac development, its role in cardiac pathology is unclear. Previous studies have shown the detrimental effects on the heart of sustained germline calreticulin overexpression, yet without calreticulin, the heart does not develop normally. Thus, carefully balanced calreticulin levels are required for the heart to develop and to function properly into adulthood. But what happens to calreticulin levels, and how is this regulated, during cardiac hypertrophy, during which the fetal gene program is reactivated, at least partially? Our working hypothesis was that c-Src, a kinase whose activity we previously found to be correlated with calreticulin expression, was involved with calreticulin in regulating the response to hypertrophic signals. Thus, we subjected adult mice to transverse aortic constriction to induce left ventricular hypertrophy. We found that aortic constriction caused calreticulin levels to increase, whereas those of c-Src fell with longer constriction time. We also examined the ability of embryonic stem cell-derived cardiomyocytes to respond to soluble hypertrophic agonists. Endothelin-1 treatment caused a significantly greater cell area increase of calreticulin-null cardiomyocytes, which had higher c-Src activity, compared with wild-type cells. c-Src inhibition abolished this difference. Greater c-Src activity may explain the efficacy with which calreticulin-null cells are able to induce the hypertrophic program, while cells containing calreticulin may be able to attenuate the hypertrophic response as a result of decreased c-Src activity. Thus, calreticulin may have a protective effect on the heart in the face of cardiac hypertrophy.

Calreticulin promotes cell motility and enhances resistance to anoikis through STAT3-CTTN-Akt pathway in esophageal squamous cell carcinoma.

We have shown earlier that overexpression of Calreticulin (CRT) contributed to a poor prognosis for patients with esophageal squamous cell carcinoma (ESCC). Here, we have shown an important role of CRT in tumorigenesis through enhancing cell motility and anoikis resistance. SiRNA-mediated knockdown of CRT caused impaired cell migration, invasion and resistance to anoikis. Notably, CRT downregulation decreased the expression of Cortactin (CTTN), which has been previously reported as a candidate oncogene associated with anoikis through the PI3K-Akt pathway. In addition, Akt phosphorylation was abolished after CRT downregulation and its activation can be refreshed by CRT upregulation, suggesting that CRT-enhanced cell resistance to anoikis through the CRT-CTTN-PI3K-Akt pathway. Moreover, the CTTN mRNA level was decreased in CRT-siRNA cells, coupled with the inactivation of STAT3. Expression of both CTTN and p-STAT3 was reduced in tumor cells following incubation with the JAK-specific inhibitor, AG490. Chromatin immunoprecipitation assay showed direct binding of p-STAT3 to the conservative STAT3-binding sequences in CTTN promoter. Furthermore, overexpression of CTTN in CRT-downregulated ESCC cells restored its motility and resistance to anoikis. This study not only reveals a role of CRT in motility promotion and anoikis resistance in ESCC cells, but also identifies CRT as an upstream regulator in the CRT-STAT3-CTTN-Akt pathway.

Caenorhabditis elegans glutamate transporter deletion induces AMPA-receptor/adenylyl cyclase 9-dependent excitotoxicity.

In stroke and several neurodegenerative diseases, malfunction of glutamate (Glu) transporters causes Glu accumulation and triggers excitotoxicity. Many details on the cascade of events in the neurodegenerative process remain unclear. As molecular components of glutamatergic synapses are assembled in Caenorhabditis elegans and as many fundamental cellular processes are conserved from nematodes to humans, we studied Glu-induced necrosis in C. elegans and probed its genetic requirements. We combined deltaglt-3, a Glu transporter-null mutation, with expression of a constitutively active form of the alpha subunit of the G protein Gs. While neither deltaglt-3 nor expression of the constitutively active form of the alpha subunit of the G protein Gs is severely toxic to C. elegans head interneurons, their combination induces extensive neurodegeneration. deltaglt-3-dependent neurodegeneration acts through Ca2+-permeable Glu receptors of the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, requires calreticulin function, and is modulated by calcineurin and type-9 adenylyl cyclase (AC9). We further show that mammalian AC9 hyperactivates mammalian AMPA-receptors (AMPA-Rs) in a Xenopus oocyte expression system, supporting that the relationship between AMPA-Rs hyperactivation and AC9 might be conserved between nematodes and mammals. AMPA-Rs-AC9 synergism is thus critical for nematode excitotoxicity and could potentially be involved in some forms of mammalian neurodegeneration.

Calreticulin inhibits commitment to adipocyte differentiation.

Calreticulin, an endoplasmic reticulum (ER) resident protein, affects many critical cellular functions, including protein folding and calcium homeostasis. Using embryonic stem cells and 3T3-L1 preadipocytes, we show that calreticulin modulates adipogenesis. We find that calreticulin-deficient cells show increased potency for adipogenesis when compared with wild-type or calreticulin-overexpressing cells. In the highly adipogenic crt(-/-) cells, the ER lumenal calcium concentration was reduced. Increasing the ER lumenal calcium concentration led to a decrease in adipogenesis. In calreticulin-deficient cells, the calmodulin-Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) pathway was up-regulated, and inhibition of CaMKII reduced adipogenesis. Calreticulin inhibits adipogenesis via a negative feedback mechanism whereby the expression of calreticulin is initially up-regulated by peroxisome proliferator-activated receptor gamma (PPAR gamma). This abundance of calreticulin subsequently negatively regulates the expression of PPAR gamma, lipoprotein lipase, CCAAT enhancer-binding protein alpha, and aP2. Thus, calreticulin appears to function as a Ca(2+)-dependent molecular switch that regulates commitment to adipocyte differentiation by preventing the expression and transcriptional activation of critical proadipogenic transcription factors.

Inhibition of adipogenesis: a new job for the ER Ca2+ pool.

Adipogenesis is the process of differentiation of adipocytes from mesenchymal multipotent cells through adipocyte precursors. In this issue, a study by the groups of Opas and Michalak (Szabo, E., Y. Qiu, S. Baksh, M. Michalak, and M. Opas. 2008. J. Cell. Biol. 182:103-116) demonstrates that this process is repressed by increasing intracellular Ca(2+), which, in turn, is dependent on the expression of calreticulin, the major Ca(2+)-binding protein of the endoplasmic reticulum lumen.

Lectin-deficient calreticulin retains full functionality as a chaperone for class I histocompatibility molecules.

Calreticulin is a molecular chaperone of the endoplasmic reticulum that uses both a lectin site specific for Glc(1)Man(5-9)GlcNAc(2) oligosaccharides and a polypeptide binding site to interact with nascent glycoproteins. The latter mode of substrate recognition is controversial. To examine the relevance of polypeptide binding to protein folding in living cells, we prepared lectin-deficient mutants of calreticulin and examined their abilities to support the assembly and quality control of mouse class I histocompatibility molecules. In cells lacking calreticulin, class I molecules exhibit inefficient loading of peptide ligands, reduced cell surface expression and aberrantly rapid export from the endoplasmic reticulum. Remarkably, expression of calreticulin mutants that are completely devoid of lectin function fully complemented all of the class I biosynthetic defects. We conclude that calreticulin can use nonlectin-based modes of substrate interaction to effect its chaperone and quality control functions on class I molecules in living cells. Furthermore, pulse-chase coimmunoisolation experiments revealed that lectin-deficient calreticulin bound to a similar spectrum of client proteins as wild-type calreticulin and dissociated with similar kinetics, suggesting that lectin-independent interactions are commonplace in cells and that they seem to be regulated during client protein maturation.

Kinase-dependent adhesion to fibronectin: regulation by calreticulin.

We studied the phosphorylation (activation status) of c-Src and CaMKII in MEFs either wild type for calreticulin, calreticulin-null, or rescued with full-length calreticulin. We found that calreticulin-null cells were poorly spread on the substratum and formed few, if any, focal contacts. Fibronectin expression and deposition were lower in calreticulin-null MEFs compared to calreticulin-expressing cells, which also exhibited increased c-Src and CaMKII phosphorylation (activity). Plating MEFs on preformed fibronectin rescued the poor adhesive phenotype of calreticulin-null cells, and caused a decrease in c-Src Y418 phosphorylation (activity). c-Src inhibition caused the calreticulin-null MEFs to become well spread on the substratum and to make many prominent focal contacts. Calmodulin and CaMKII inhibition caused similar results, along with a notable increase in paxillin phosphorylation (activation). To test if the calcium storage function of calreticulin was responsible for these effects, we manipulated intracellular [Ca(2+)]. Lowering [Ca(2+)](ER) caused an increase in c-Src phosphorylation and a decrease in fibronectin abundance. Conversely, increasing [Ca(2+)] caused opposite effects. These results suggest that calreticulin regulates both the c-Src and calmodulin/CaMKII pathways, enabling cells to be better spread on the substratum by allowing greater fibronectin deposition and increased focal contact formation.

Differential expression and activity of matrix metalloproteinase-2 and -9 in the calreticulin deficient cells.

Calreticulin is an endoplasmic reticulum protein important in cardiovascular development. Deletion of the calreticulin gene leads to defects in the heart and the formation of omphaloceal. These defects could both be due to changes in the extracellular matrix composition. Matrix metalloproteinases (MMP)-2 and MMP-9 are two of the MMPs which are essential for cardiovascular remodelling and development. Here, we tested the hypothesis that the defects observed in the heart and body wall of the calreticulin null embryos are due to alterations in MMP-2 and MMP-9 activity. Our results demonstrate that there is a significant decrease in the MMP-9 and increase in the MMP-2 activity and expression in the calreticulin deficient cells. We also showed that there is a significant increase in the expression level of membrane type-1 matrix metalloproteinase (MT1-MMP). In contrast, there was no change in the tissue inhibitor of matrix metalloproteinase (TIMP)-1 or -2 in the calreticulin deficient cells as compared to the wild type cells. Interestingly, the inhibition of the MEK kinase pathway using PD98059 attenuated the decrease in the MMP-9 mRNA with no effect on the MMP-2 mRNA level in the calreticulin deficient cells. Furthermore, PI3 kinase inhibitor decreased the expression of both the MMP-2 and MMP-9. This study is the first report on the role of calreticulin in regulating MMP activity.

Ultrastructural analysis of development of myocardium in calreticulin-deficient mice.

BACKGROUND: Calreticulin is a Ca2+ binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours. RESULTS: We show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism. CONCLUSION: Calreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene.