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Calreticulin in cardiac development and pathology
Biochimica et Biophysica Acta 1600 (2002) 32 – 37 www.bba-direct.com
Review
Calreticulin in cardiac development and pathology Marek Michalak a,*, Jeffrey Lynch a, Jody Groenendyk a, Lei Guo a, J.M. Robert Parker a, Michal Opas b a
Canadian Institutes of Health Research Membrane Protein Research Group and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 b Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Received 15 June 2002; accepted 19 September 2002
Abstract Calreticulin is a Ca2 + binding/storage chaperone resident in the lumen of endoplasmic reticulum (ER). The protein is an important component of the calreticulin/calnexin cycle and the quality control pathways in the ER. In mice, calreticulin deficiency is lethal due to impaired cardiac development. This is not surprising because the protein is expressed at high level at early stages of cardiac development. Overexpression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Recent studies on calreticulin-deficient and transgenic mice revealed that the protein is a key upstream regulator of calcineurin-dependent pathways during cardiac development. Calreticulin and ER may play important role in cardiac development and postnatal pathologies. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Endoplasmic reticulum; Chaperone; Calcium binding protein; Cardiac development; Cardiac arrhythmia
1. Introduction Endoplasmic reticulum (ER) plays a vital role in many cellular processes including Ca2 + storage and release, lipid synthesis, protein synthesis, folding and posttranslational modification [1]. Lumen of the ER contains a large concentration of resident proteins involved in virtually all aspects of ER functions [1– 9]. Many severe protein folding diseases result from impaired function of the ER membrane and its luminal protein folding machinery [9– 12]. The ER also contains a high concentration of Ca2 +, most of it buffered bound to ER resident chaperones [4,6]. Significant portion of ER Ca2 + is free and it affects many Ca2 +dependent processes in the ER lumen [4,6]. Not surprisingly, fluctuations of the ER luminal Ca2 + concentration modulate quality and efficiency of protein folding [6,13 – 15]. Ca2 + stored in the ER lumen is also released via inositol 1,4,5-trisphosphate (InsP3)-receptor pathway to
Abbreviations: ER, endoplasmic reticulum; InsP3, inositol 1,4,5trisphosphate; NF-AT, nuclear factor of activated T-cells * Corresponding author. Tel.: +1-780-492-2256; fax: +1-780-492-0886. E-mail address: [email protected] (M. Michalak).
activate many important Ca2 +-dependent processes in the cytoplasmic compartment [16]. Calreticulin is a major Ca2 +-binding chaperone residing in the ER lumen [17]. The protein binds (buffers) Ca2 + with high capacity and it participates in folding of newly synthesized proteins and glycoproteins [17 –19]. Recently, studies on calreticulin-deficient and calreticulin transgenic mice revealed that the protein plays a vital role in cardiac physiology and pathologies. In this review, we focused on the role of calreticulin in cardiac development and postnatal pathologies.
2. Calreticulin, a Ca2+-binding chaperone Several years of structural and functional studies indicate that calreticulin may be divided into distinct regions (domains) [17]. Fig. 1 shows a putative model of calreticulin domains including detailed structure of the central P-domain of the protein as revealed by latest NMR studies [20]. The globular N-domain of calreticulin has been modeled based on the available crystallographic data reported for calnexin [21]. This was possible because of a great degree of amino acid sequence identity and functional similarities between calreticulin and calnexin [2]. The P-domain of calreticulin ([20],
1570-9639/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 7 0 - 9 6 3 9 ( 0 2 ) 0 0 4 4 1 - 7
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Fig. 1. A putative model of calreticulin domains. (A) shows schematic representation of calreticulin domains considering that central, proline-rich P-domain of the protein forms a characteristic loop formed [20]. The locations of repeats 1 (-I-x-D-P-(D/E)-A-x-K-P-E-D-W-D-(D/E)-) and 2 (-G-x-W-x-x-P-x-I-N-x-P-x-Y-) in the P-domain is indicated. In (B) the N- and P-domain of calreticulin are modeled based on he NMR studies of the P-domain of calreticulin [20] and crystallographic studies of calnexin (1JHN) [21]. The amino acid sequence alignment of calreticulin and calnexin was adjusted such that insertions and deletions were in loop regions. The alignment was started with residue E1 of calreticulin residue E1 and S67 of calnexin. The globular N-domain is shown in green and the extended arm of the P-domain is represented in red. Colored balls represent the cysteine (Cys88 – Cys120), which form a C – C bridge in calreticulin. Putative glucose (carbohydrate?) binding site is depicted in a similar localization of glucose binding site in calnexin [21]. There is no structural information for the C-domain shown as a blue cylinder. The N + P-domain is likely a chaperone region of calreticulin, whereas the C-domain is responsible for Ca2 + storage function in the ER lumen [25].
Fig. 1) and calnexin [21] has a highly unusual structure. It forms an extended and curved arm connected to a globular Ndomain. The elongated arm of the P-domain provides a site of attachment for other chaperones including ERp57 [22]. The N-terminal globular domain likely forms anti-parallel hsheets as shown for calnexin [21] and as predicted earlier from the amino acid sequence of calreticulin [23,24]. The exact 3D structure of the N- and C-domain of calreticulin will require further crystallographic and NMR studies. However, a model of calreticulin presented in Fig. 1 should be useful to design future structural/functional studies on the protein. As
indicated in Fig. 1, the N-terminal region of the protein, encompassing the N- and P-domain of calreticulin, is involved in chaperone function of the protein [18,22,25] (Fig. 1). It interacts with misfolded proteins and glycoproteins, binds ATP, Zn2 + and Ca2 + with high affinity and low capacity, all likely involved in the chaperone function of calreticulin [18,26 –31] (Fig. 1). The C-terminal region of calreticulin binds Ca2 + with high capacity and plays a role in Ca2 + storage in the lumen of the ER in vivo [25] (Fig. 1). Unfortunately, at present, there are no structural information available on the acidic C-domain of the proteins.
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Many unique functions have been reported for calreticulin, including modulation of Ca2 + homeostasis, chaperone activity, regulation of cell adhesion and of gene expression, regulation of nuclear export to name a few [2,25,32 – 44]. Here we focus on the role of calreticulin in protein folding and modulation of Ca2 + homeostasis and its implication to cardiac physiology and pathology. Role of calreticulin in Ca2 + binding and regulation of Ca homeostasis has been extensively studied and very well documented [17,25,27, 37 –40,43,45]. For example, overexpression of calreticulin results in an increased amount of intracellularly stored Ca2 + [37,39,41,43], whereas, calreticulin-deficient cells have reduced ER Ca2 + storage capacity [25]. Calreticulin-deficient cells have impaired InsP3-dependent Ca2 + release [45]. Store-operated Ca2 + influx is also altered in cells overexpressing calreticulin [37,39,41,43,46]. Molecular mechanisms responsible for calreticulin-dependent effects on the store-operated Ca2 + influx are not well understood. Ca2 + binding to calreticulin and, consequently, changes in the ER Ca2 + capacity, affect chaperone function of the protein and influence ‘‘quality control’’ of the secretory pathway [6,13 – 15,30,31]. It is very well documented that calreticulin also functions as a molecular chaperone for many proteins and glycoproteins [2,18,32]. The protein binds monoglucosylated oligosaccharides [47 – 49] and
binds to nonglycosylated proteins [18]. The N-terminal globular region of the protein, similarly to calnexin [21], binds carbohydrates [49] and plays a role in chaperone function of calreticulin [25] (Fig. 1).
3. Calreticulin, a new cardiac embryonic gene Calreticulin-deficient mice die because of impaired heart development [45,50]. This was initially a surprising finding because the protein is only a minor component of cardiac cells [23,45,51– 54]. However, careful analysis of activation patterns of the calreticulin promoter in transgenic animals revealed that the calreticulin gene is highly active in the developing heart [45]. This suggested that calreticulin, a Ca2 +-binding chaperone of the ER, is a novel cardiac embryonic gene [45]. Mouse embryonic fibroblasts isolated from calreticulindeficient embryos have impaired agonist-induced Ca2 + release and inhibited nuclear import of NF-ATc1 transcription factor, suggesting that calreticulin may control the availability of Ca2 + ions required for activation of calcineurin-dependent nuclear translocation of NF-ATc1 during cardiac development [25,45]. Indeed, activation of the NFAT/GATA-4/calcineurin transcriptional pathway depends on
Fig. 2. Relationship between calreticulin in the ER lumen and Ca2 +-dependent transcriptional pathways in developing cardiomyocytes. Calreticulin, in the lumen of the ER, plays a role of Ca2 +-binding/storage chaperone. The protein affects Ca2 + capacity of the ER stores and plays a critical role in the folding of many proteins and glycoproteins including plasma membrane channels, transporters and receptors. Red arrows indicate calreticulin-sensitive pathways, which are inhibited in calreticulin-deficient cells [25,45,74]. A relationship between calreticulin, Ca2 +, calcineurin and NF-AT is emphasized. CRT, calreticulin; CaN, calcineurin, InsP3R, InsP3 receptor; SERCA, sarcoplasmic, endoplasmic reticulum Ca2 +-ATPase; NF-AT, nuclear factor of activated T-cells.
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Ca2 + release from the ER and Ca2 + entry via plasma membrane Ca2 + channels [45,55 – 59]. Therefore, cardiac muscle must contain InsP3-sensitive, ER-like Ca2 + storage compartment functionally distinct from the sarcoplasmic reticulum [60 –62]. Studies on calreticulin-deficient cell lines revealed that their bradykinin-induced Ca2 + release is impaired likely due to the inability of the bradykinin receptor to bind its ligand with high affinity [25,45]. Thrombin receptor is also nonfunctional because thrombin-induced Ca2 + release from the ER is inhibited in calreticulin-deficient cells (unpublished observations). These findings led to a hypothesis that calreticulin-deficient cells have inhibited activity of calcineurin and consequently inhibited cardiac specific transcriptional processes at the early stages of cardiac development [45]. Fig. 2 shows a schematic representation of relationship between calreticulin in the ER lumen and Ca2 +-dependent transcriptional processes. Of special interest is an association between calreticulin- and calcineurin-dependent processes. Calcineurin is a serine/threonine phosphatase, which participates in many signaling pathways [63 – 65] and it consists of catalytic (A) and regulatory (B) subunits [63,65]. A Ca2 +- and calcineurin B-dependent regulatory region is located in the C-terminal 200 amino acids of the catalytic subunit [63,65]. But deletion of the C-terminal 200 amino acid residues from the protein, yields a fully active and constitutive phosphatase [66]. Remarkably, overexpression of activated calcineurin reverses the defect in cardiac development observed in calreticulin-deficient mice and rescues them from embryonic lethality (unpublished). This indicates that a constitutively active Ca2 +-independent variant of calcineurin permits the proper progression of cardiac development during embryogenesis in the absence of calreticulin. This fully supports our hypothesis that Ca2 +dependent cardiac specific transcriptional processes are impaired in the absence of calreticulin [45] and it shows that calreticulin must be the key upstream molecule to calcineurin and calcineurin-dependent transcriptional events during cardiac development.
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complete AV nodal conduction block. These findings indicate that calreticulin may play a role in the pathology and development of the conductive system [68]. Furthermore, calreticulin overexpresser hearts have very low levels of Connexin43 [68], a major component of gap junctions responsible for cell – cell communication [69,70]. The current density of peak inward ICa,L at 0 mV elicited in transgenic cardiomyocytes was also significantly smaller than the ICa,L of control animals under the same condition [68]. Recent studies on H9c2 cells indicate that overexpression of calreticulin in cardiomyocytes affects the Akt signaling pathway and promotes apoptosis [71]. Full understanding of the molecular events responsible for calreticulin-dependent conductive pathology will require further investigations. The most significant finding is that the phenotype of the calreticulin overexpresser mouse is very similar to that seen in children with complete heart block [68,72,73]. The cause and molecular mechanism involved in the complete heart block is not known at present. However, calreticulin must be part of a pathway responsible for the etiology of this disease. The protein may affect Ca2 + homeostasis in differentiating cardiomyocytes and it may play a critical role during synthesis, folding and targeting of Ca2 + channels, connexins and perhaps other cardiac specific proteins.
5. Conclusions It is now evident that calreticulin is a new cardiac embryonic gene. The protein is highly expressed in embryonic heart and it is essential during cardiac development. Latest work indicates that calreticulin plays an important role upstream of calcineurin-dependent transcriptional processes during cardiac development. The role of calreticulin as a modulator of Ca2 + homeostasis is likely important in early stages of cardiac development. In contrast, postnatally high level of expression of calreticulin leads to impaired development of the cardiac conductive system and may be responsible for the pathology of the complete heart block.
4. Calreticulin and congenital complete heart block Acknowledgements Calreticulin expression is high in embryonic heart and declines sharply after birth likely due to activation of the gene by Nkx2.5 transcription factor and its suppression by COUP-TF1 [67]. Why is the expression of calreticulin so tightly regulated in the heart? To answer this intriguing question, transgenic mice have recently been created that overexpress calreticulin in the heart [68]. These animals develop bradycardia associated with sinus node dysfunction, complete cardiac block, and death due to intractable heart failure. Electrocardiograms demonstrated that the P – R interval of transgenic mice is significantly prolonged in calreticulin overexpressers with subsequent development of
We thank Michel Puceat (CNRS Montpellier) for help during preparation of this manuscript. Work in our laboratories is supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario. J.L. is a recipient of a Studentship from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research. J.G. is a recipient of a Studentship from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research. L.G. is a postdoctoral fellow of the Heart and Stroke Foundation of Canada. M.M. is a Canadian Institutes of
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Health Research Senior Investigator and a Medical Scientist of the Alberta Heritage Foundation for Medical Research.
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Review
Calreticulin in cardiac development and pathology Marek Michalak a,*, Jeffrey Lynch a, Jody Groenendyk a, Lei Guo a, J.M. Robert Parker a, Michal Opas b a
Canadian Institutes of Health Research Membrane Protein Research Group and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 b Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Received 15 June 2002; accepted 19 September 2002
Abstract Calreticulin is a Ca2 + binding/storage chaperone resident in the lumen of endoplasmic reticulum (ER). The protein is an important component of the calreticulin/calnexin cycle and the quality control pathways in the ER. In mice, calreticulin deficiency is lethal due to impaired cardiac development. This is not surprising because the protein is expressed at high level at early stages of cardiac development. Overexpression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Recent studies on calreticulin-deficient and transgenic mice revealed that the protein is a key upstream regulator of calcineurin-dependent pathways during cardiac development. Calreticulin and ER may play important role in cardiac development and postnatal pathologies. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Endoplasmic reticulum; Chaperone; Calcium binding protein; Cardiac development; Cardiac arrhythmia
1. Introduction Endoplasmic reticulum (ER) plays a vital role in many cellular processes including Ca2 + storage and release, lipid synthesis, protein synthesis, folding and posttranslational modification [1]. Lumen of the ER contains a large concentration of resident proteins involved in virtually all aspects of ER functions [1– 9]. Many severe protein folding diseases result from impaired function of the ER membrane and its luminal protein folding machinery [9– 12]. The ER also contains a high concentration of Ca2 +, most of it buffered bound to ER resident chaperones [4,6]. Significant portion of ER Ca2 + is free and it affects many Ca2 +dependent processes in the ER lumen [4,6]. Not surprisingly, fluctuations of the ER luminal Ca2 + concentration modulate quality and efficiency of protein folding [6,13 – 15]. Ca2 + stored in the ER lumen is also released via inositol 1,4,5-trisphosphate (InsP3)-receptor pathway to
Abbreviations: ER, endoplasmic reticulum; InsP3, inositol 1,4,5trisphosphate; NF-AT, nuclear factor of activated T-cells * Corresponding author. Tel.: +1-780-492-2256; fax: +1-780-492-0886. E-mail address: [email protected] (M. Michalak).
activate many important Ca2 +-dependent processes in the cytoplasmic compartment [16]. Calreticulin is a major Ca2 +-binding chaperone residing in the ER lumen [17]. The protein binds (buffers) Ca2 + with high capacity and it participates in folding of newly synthesized proteins and glycoproteins [17 –19]. Recently, studies on calreticulin-deficient and calreticulin transgenic mice revealed that the protein plays a vital role in cardiac physiology and pathologies. In this review, we focused on the role of calreticulin in cardiac development and postnatal pathologies.
2. Calreticulin, a Ca2+-binding chaperone Several years of structural and functional studies indicate that calreticulin may be divided into distinct regions (domains) [17]. Fig. 1 shows a putative model of calreticulin domains including detailed structure of the central P-domain of the protein as revealed by latest NMR studies [20]. The globular N-domain of calreticulin has been modeled based on the available crystallographic data reported for calnexin [21]. This was possible because of a great degree of amino acid sequence identity and functional similarities between calreticulin and calnexin [2]. The P-domain of calreticulin ([20],
1570-9639/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 7 0 - 9 6 3 9 ( 0 2 ) 0 0 4 4 1 - 7
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Fig. 1. A putative model of calreticulin domains. (A) shows schematic representation of calreticulin domains considering that central, proline-rich P-domain of the protein forms a characteristic loop formed [20]. The locations of repeats 1 (-I-x-D-P-(D/E)-A-x-K-P-E-D-W-D-(D/E)-) and 2 (-G-x-W-x-x-P-x-I-N-x-P-x-Y-) in the P-domain is indicated. In (B) the N- and P-domain of calreticulin are modeled based on he NMR studies of the P-domain of calreticulin [20] and crystallographic studies of calnexin (1JHN) [21]. The amino acid sequence alignment of calreticulin and calnexin was adjusted such that insertions and deletions were in loop regions. The alignment was started with residue E1 of calreticulin residue E1 and S67 of calnexin. The globular N-domain is shown in green and the extended arm of the P-domain is represented in red. Colored balls represent the cysteine (Cys88 – Cys120), which form a C – C bridge in calreticulin. Putative glucose (carbohydrate?) binding site is depicted in a similar localization of glucose binding site in calnexin [21]. There is no structural information for the C-domain shown as a blue cylinder. The N + P-domain is likely a chaperone region of calreticulin, whereas the C-domain is responsible for Ca2 + storage function in the ER lumen [25].
Fig. 1) and calnexin [21] has a highly unusual structure. It forms an extended and curved arm connected to a globular Ndomain. The elongated arm of the P-domain provides a site of attachment for other chaperones including ERp57 [22]. The N-terminal globular domain likely forms anti-parallel hsheets as shown for calnexin [21] and as predicted earlier from the amino acid sequence of calreticulin [23,24]. The exact 3D structure of the N- and C-domain of calreticulin will require further crystallographic and NMR studies. However, a model of calreticulin presented in Fig. 1 should be useful to design future structural/functional studies on the protein. As
indicated in Fig. 1, the N-terminal region of the protein, encompassing the N- and P-domain of calreticulin, is involved in chaperone function of the protein [18,22,25] (Fig. 1). It interacts with misfolded proteins and glycoproteins, binds ATP, Zn2 + and Ca2 + with high affinity and low capacity, all likely involved in the chaperone function of calreticulin [18,26 –31] (Fig. 1). The C-terminal region of calreticulin binds Ca2 + with high capacity and plays a role in Ca2 + storage in the lumen of the ER in vivo [25] (Fig. 1). Unfortunately, at present, there are no structural information available on the acidic C-domain of the proteins.
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Many unique functions have been reported for calreticulin, including modulation of Ca2 + homeostasis, chaperone activity, regulation of cell adhesion and of gene expression, regulation of nuclear export to name a few [2,25,32 – 44]. Here we focus on the role of calreticulin in protein folding and modulation of Ca2 + homeostasis and its implication to cardiac physiology and pathology. Role of calreticulin in Ca2 + binding and regulation of Ca homeostasis has been extensively studied and very well documented [17,25,27, 37 –40,43,45]. For example, overexpression of calreticulin results in an increased amount of intracellularly stored Ca2 + [37,39,41,43], whereas, calreticulin-deficient cells have reduced ER Ca2 + storage capacity [25]. Calreticulin-deficient cells have impaired InsP3-dependent Ca2 + release [45]. Store-operated Ca2 + influx is also altered in cells overexpressing calreticulin [37,39,41,43,46]. Molecular mechanisms responsible for calreticulin-dependent effects on the store-operated Ca2 + influx are not well understood. Ca2 + binding to calreticulin and, consequently, changes in the ER Ca2 + capacity, affect chaperone function of the protein and influence ‘‘quality control’’ of the secretory pathway [6,13 – 15,30,31]. It is very well documented that calreticulin also functions as a molecular chaperone for many proteins and glycoproteins [2,18,32]. The protein binds monoglucosylated oligosaccharides [47 – 49] and
binds to nonglycosylated proteins [18]. The N-terminal globular region of the protein, similarly to calnexin [21], binds carbohydrates [49] and plays a role in chaperone function of calreticulin [25] (Fig. 1).
3. Calreticulin, a new cardiac embryonic gene Calreticulin-deficient mice die because of impaired heart development [45,50]. This was initially a surprising finding because the protein is only a minor component of cardiac cells [23,45,51– 54]. However, careful analysis of activation patterns of the calreticulin promoter in transgenic animals revealed that the calreticulin gene is highly active in the developing heart [45]. This suggested that calreticulin, a Ca2 +-binding chaperone of the ER, is a novel cardiac embryonic gene [45]. Mouse embryonic fibroblasts isolated from calreticulindeficient embryos have impaired agonist-induced Ca2 + release and inhibited nuclear import of NF-ATc1 transcription factor, suggesting that calreticulin may control the availability of Ca2 + ions required for activation of calcineurin-dependent nuclear translocation of NF-ATc1 during cardiac development [25,45]. Indeed, activation of the NFAT/GATA-4/calcineurin transcriptional pathway depends on
Fig. 2. Relationship between calreticulin in the ER lumen and Ca2 +-dependent transcriptional pathways in developing cardiomyocytes. Calreticulin, in the lumen of the ER, plays a role of Ca2 +-binding/storage chaperone. The protein affects Ca2 + capacity of the ER stores and plays a critical role in the folding of many proteins and glycoproteins including plasma membrane channels, transporters and receptors. Red arrows indicate calreticulin-sensitive pathways, which are inhibited in calreticulin-deficient cells [25,45,74]. A relationship between calreticulin, Ca2 +, calcineurin and NF-AT is emphasized. CRT, calreticulin; CaN, calcineurin, InsP3R, InsP3 receptor; SERCA, sarcoplasmic, endoplasmic reticulum Ca2 +-ATPase; NF-AT, nuclear factor of activated T-cells.
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Ca2 + release from the ER and Ca2 + entry via plasma membrane Ca2 + channels [45,55 – 59]. Therefore, cardiac muscle must contain InsP3-sensitive, ER-like Ca2 + storage compartment functionally distinct from the sarcoplasmic reticulum [60 –62]. Studies on calreticulin-deficient cell lines revealed that their bradykinin-induced Ca2 + release is impaired likely due to the inability of the bradykinin receptor to bind its ligand with high affinity [25,45]. Thrombin receptor is also nonfunctional because thrombin-induced Ca2 + release from the ER is inhibited in calreticulin-deficient cells (unpublished observations). These findings led to a hypothesis that calreticulin-deficient cells have inhibited activity of calcineurin and consequently inhibited cardiac specific transcriptional processes at the early stages of cardiac development [45]. Fig. 2 shows a schematic representation of relationship between calreticulin in the ER lumen and Ca2 +-dependent transcriptional processes. Of special interest is an association between calreticulin- and calcineurin-dependent processes. Calcineurin is a serine/threonine phosphatase, which participates in many signaling pathways [63 – 65] and it consists of catalytic (A) and regulatory (B) subunits [63,65]. A Ca2 +- and calcineurin B-dependent regulatory region is located in the C-terminal 200 amino acids of the catalytic subunit [63,65]. But deletion of the C-terminal 200 amino acid residues from the protein, yields a fully active and constitutive phosphatase [66]. Remarkably, overexpression of activated calcineurin reverses the defect in cardiac development observed in calreticulin-deficient mice and rescues them from embryonic lethality (unpublished). This indicates that a constitutively active Ca2 +-independent variant of calcineurin permits the proper progression of cardiac development during embryogenesis in the absence of calreticulin. This fully supports our hypothesis that Ca2 +dependent cardiac specific transcriptional processes are impaired in the absence of calreticulin [45] and it shows that calreticulin must be the key upstream molecule to calcineurin and calcineurin-dependent transcriptional events during cardiac development.
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complete AV nodal conduction block. These findings indicate that calreticulin may play a role in the pathology and development of the conductive system [68]. Furthermore, calreticulin overexpresser hearts have very low levels of Connexin43 [68], a major component of gap junctions responsible for cell – cell communication [69,70]. The current density of peak inward ICa,L at 0 mV elicited in transgenic cardiomyocytes was also significantly smaller than the ICa,L of control animals under the same condition [68]. Recent studies on H9c2 cells indicate that overexpression of calreticulin in cardiomyocytes affects the Akt signaling pathway and promotes apoptosis [71]. Full understanding of the molecular events responsible for calreticulin-dependent conductive pathology will require further investigations. The most significant finding is that the phenotype of the calreticulin overexpresser mouse is very similar to that seen in children with complete heart block [68,72,73]. The cause and molecular mechanism involved in the complete heart block is not known at present. However, calreticulin must be part of a pathway responsible for the etiology of this disease. The protein may affect Ca2 + homeostasis in differentiating cardiomyocytes and it may play a critical role during synthesis, folding and targeting of Ca2 + channels, connexins and perhaps other cardiac specific proteins.
5. Conclusions It is now evident that calreticulin is a new cardiac embryonic gene. The protein is highly expressed in embryonic heart and it is essential during cardiac development. Latest work indicates that calreticulin plays an important role upstream of calcineurin-dependent transcriptional processes during cardiac development. The role of calreticulin as a modulator of Ca2 + homeostasis is likely important in early stages of cardiac development. In contrast, postnatally high level of expression of calreticulin leads to impaired development of the cardiac conductive system and may be responsible for the pathology of the complete heart block.
4. Calreticulin and congenital complete heart block Acknowledgements Calreticulin expression is high in embryonic heart and declines sharply after birth likely due to activation of the gene by Nkx2.5 transcription factor and its suppression by COUP-TF1 [67]. Why is the expression of calreticulin so tightly regulated in the heart? To answer this intriguing question, transgenic mice have recently been created that overexpress calreticulin in the heart [68]. These animals develop bradycardia associated with sinus node dysfunction, complete cardiac block, and death due to intractable heart failure. Electrocardiograms demonstrated that the P – R interval of transgenic mice is significantly prolonged in calreticulin overexpressers with subsequent development of
We thank Michel Puceat (CNRS Montpellier) for help during preparation of this manuscript. Work in our laboratories is supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario. J.L. is a recipient of a Studentship from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research. J.G. is a recipient of a Studentship from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research. L.G. is a postdoctoral fellow of the Heart and Stroke Foundation of Canada. M.M. is a Canadian Institutes of
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Health Research Senior Investigator and a Medical Scientist of the Alberta Heritage Foundation for Medical Research.
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