A role for in the adult heart?

Alexander Maass, Leslie A. Leinwand

J Clin Invest. 2001;107(10):1223-1225. https://doi.org/10.1172/JCI13125.

Commentary

The essential role of calcium in cardiac development, function, and disease is incontrovertible. The multiple pathways that regulate Ca2+ homeostasis in the heart are complex and frequently intersecting, involving a large number of myocyte . One that is emerging as having a central role in Ca2+ homeostasis and in cardiac development is the ubiquitous protein of the (ER), calreticulin. Over 40 different functions have been ascribed to this Ca2+-binding protein, including regulation of expression, protein folding, cell adhesion, and autoimmunity (reviewed in ref. 1). In the mouse heart, calreticulin expression is activated at 9.5 days postcoitum (dpc) and remains high until 14.5 dpc. Expression is relatively low by 18 dpc and is barely detectable postnatally. Mice in which the Calreticulin gene has been inactivated die by 14.5 dpc, primarily due to cardiac defects with a marked decrease in ventricular wall thickness (2). Therefore, the importance of calreticulin in cardiac development has been solidified, although its specific role remains unclear. In this issue of JCI, Nakamura and colleagues (3) have forced expression of calreticulin in adult cardiac myocytes in the mouse, to study the sequelae of sustained high expression of this protein after development. These animals exhibit decreased systolic function, chamber dilation, and wall thinning, and they die of sudden cardiac death (3). This premature death […]

Find the latest version: https://jci.me/13125/pdf A role for calreticulin in the adult heart? Commentary

See related article, pages 1245–1253. Alexander Maass and Leslie A. Leinwand Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, USA Address correspondence to: Leslie A. Leinwand, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Campus Box 347, Boulder, Colorado 80309-0347, USA. Phone: (303) 492-7606; Fax: (303) 492-8907; E-mail: [email protected].

The essential role of calcium in car- myocytes results in severe arrhyth- These interesting findings raise issues diac development, function, and dis- mias (4). In addition, mice lacking about the potential role for calretic- ease is incontrovertible. The multiple connexin40 exhibit sinoatrial, intra- ulin in the pathogenesis of human pathways that regulate Ca2+ home- atrial, and atrioventricular conduc- arrhythmias. ostasis in the heart are complex and tion disturbances (5). The complete Several recent reviews (1, 6) have frequently intersecting, involving a heart block in calreticulin-overex- sifted through the multitude of roles large number of myocyte proteins. pressing mice might therefore be ascribed to calreticulin, seeking to One protein that is emerging as hav- explained, at least in part, by second- clarify the relevant functions of this ing a central role in Ca2+ homeostasis ary changes in expression of other protein. Calreticulin binds Ca2+ and in cardiac development is the proteins, including the connexins. through two functionally distinct ubiquitous chaperone protein of the Since other proteins that have been sites and is localized to the ER, where endoplasmic reticulum (ER), calretic- implicated in cardiac pathogenesis, it is known to function as a lectinlike ulin. Over 40 different functions have including SERCA2, , chaperone for a large number of pro- been ascribed to this Ca2+-binding , and β-myosin heavy teins. One might speculate that cal- protein, including regulation of gene chain, remain unchanged in this reticulin is involved in the proper expression, protein folding, cell adhe- model, the decrease in connexins 40 folding of these proteins, as has sion, and autoimmunity (reviewed in and 43 appears to be a relatively spe- been demonstrated for other mem- ref. 1). In the mouse heart, calretic- cific consequence of expressing the brane-associated proteins (6). How- ulin expression is activated at 9.5 days transgene. Still, reduced connexin lev- ever, the mechanism by which cal- postcoitum (dpc) and remains high els seem unlikely to explain all of its reticulin ensures proper protein until 14.5 dpc. Expression is relative- effects, since the conditional and folding and the role that Ca2+ plays in ly low by 18 dpc and is barely complete null mice for connexins 40 this process remain unknown. Given detectable postnatally. Mice in which and 43, respectively, do not exhibit that the ER is one of the largest the Calreticulin gene has been inacti- dilation and systolic dysfunction. organelles of the cell and that it is vated die by 14.5 dpc, primarily due to cardiac defects with a marked decrease in ventricular wall thickness (2). Therefore, the importance of cal- reticulin in cardiac development has been solidified, although its specific role remains unclear. In this issue of JCI, Nakamura and colleagues (3) have forced expression of calreticulin in adult cardiac myocytes in the mouse, to study the sequelae of sustained high expres- sion of this protein after develop- ment. These animals exhibit decreased systolic function, chamber dilation, and wall thinning, and they die of sudden cardiac death (3). This premature death is associated with complete heart block. While the mechanisms behind these pheno- types remain somewhat unclear, there is a significant decrease in inward Ca2+ current as well as a Figure 1 decrease in expression of connexins Involvement of calreticulin in pathways. Calreticulin is essential for integrin sig- 40 and 43. Consistent with a role for naling and the dephosphorylation and nuclear translocation of NF-AT3. The size of the Ca2+ sym- the connexins in the phenotypes of bols reflects the differing concentrations in the extracellular space and the the mice is the finding that inactiva- (10–3 M), as compared with the cytoplasm (10–5 M in systole and 10–7 M in diastole, with higher tion of connexin43 in adult cardiac levels seen in Ca2+ sparks).

The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 10 1223 arguably the most important source out at least some distinct functions cytosolic as well as localized Ca2+ con- of intracellular Ca2+, calreticulin is within the SR (12). centrations in the different cell com- well situated to play an important partments (Figure 1). This suggests a role in cardiac cell biology. Calreticulin and human heart role for Ca2+-regulating proteins in disease multiple cellular processes, such as Calreticulin deficiency and Several studies have shed light on signal transduction and cell growth. overexpression changes in several To date, there are no reports quanti- Mice with targeted disruption of the forms of heart disease, including fying calreticulin levels in human Calreticulin gene die in utero with , familial hypertrophic heart disease. decreased ventricular cell mass due to cardiomyopathy, and primary dilated Calreticulin has been implicated in increased apoptosis of cardiac cardiomyopathy (reviewed in ref. 13). congenital heart block in humans, myocytes (7). Calreticulin-deficient Changes in the expression of calcium- since autoantibodies against calretic- cells show reduced nuclear import of transporting proteins (such as ulin have been identified in a subset of the transcription factor nuclear factor SERCA and the Na+/Ca2+ exchanger) patients (19). Because the exact physio- of activated transcription 3 (NF-AT3), and their regulators (such as phos- logical function of this protein in the indicating that calreticulin partici- pholamban) have been observed in adult heart remains obscure, the pates in the Ca2+/calcineurin/NF- many forms of acquired and genetic pathophysiological relevance of AT/GATA4 signal transduction path- heart diseases, most notably in car- autoantibodies is also unclear. The way (2). Integrin-mediated Ca2+ diac hypertrophy and heart failure transgenic mice described in the pres- signaling and cell adhesion are also (reviewed in ref. 14). These changes ent work overexpress calreticulin and impaired in calreticulin-deficient cells seem to be secondary to the primary have a , but (8) (Figure 1). Mice with targeted dis- cardiac dysfunction, but recent publi- they also exhibit a complete heart ruption of GATA4 or NFATc also show cations have implicated these changes block, further obscuring calreticulin’s impairment of early cardiac develop- as major contributors to systolic and roles in these disease pathways. How- ment, but their phenotypes are dis- diastolic dysfunction. In fact, target- ever, congenital heart block and car- tinct from those of the Calreticulin ed disruption of the Phospholamban diomyopathy might share common null mice. Mice null for GATA4 fail gene seems to rescue or delay patho- molecular mechanisms, as suggested heart tube formation and die between genesis in several mouse models of by recent evidence that patients with 8.5 and 10.5 dpc (9). Mice lacking NF- dilated and hypertrophic cardiomy- congenital heart block are prone to ATc fail to form normal cardiac valves opathy (15, 16). In addition to the dilated cardiomyopathy even when and die from circulatory failure obvious role of Ca2+ in the cardiac their cardiac conduction is restored by around 14.5 dpc (10, 11). Therefore, contraction cycle, several calcium- an artificial pacemaker (20). the embryonic lethality of the calreti- dependent enzymes such as the phos- Studies with calreticulin-deficient culin-deficient mice cannot be phatase calcineurin and the family of cells suggest that this protein partici- explained simply by the disruption of Ca2+/-dependent pates in signal transduction pathways NF-AT/GATA4 pathways. have been implicated in the develop- involving integrins or calcineurin, The other main Ca2+-binding chap- ment of cardiac hypertrophy and which are thought to drive myocyte erone of the sarcoplasmic reticulum heart failure (reviewed in ref. 17). Ca2+ hypertrophy and other pathophysio- (SR) is calsequestrin, a protein that is levels change rapidly in the cytoplasm logical changes in the heart. The nor- expressed in higher amounts in the during contraction and relaxation, mal adult myocardium, however, adult heart than calreticulin. Overex- but there are also localized, subcellu- expresses only low levels of calreticulin, pression of calsequestrin in the mouse lar changes known as Ca2+ sparks raising the possibility that calreticulin heart is associated with yet another (18). Ion channels as well as calcium- is induced with the fetal gene program phenotype, involving cardiac hyper- binding proteins such as calse- that is reactivated during cardiac trophy and decreased contractility. questrin and calreticulin are impor- hypertrophy and failure. However, Hence, the two chaperones likely carry tant in the regulation of global other fetal gene products normally

Table 1 Phenotypes of transgenic mice with alterations in connexin40, connexin43, calsequestrin, or calreticulin

Gene product Overexpression (O) Conduction Sudden Effects on Arrhythmia Contractility Ref. Knockout (K) velocity cardiac death gene expression Calreticulin O (heart) ↓↓↓ + Cx43, Cx40 ↓↓ Complete heart ↓ 3 ANF, BNP ↑ block Calsequestrin O (heart) ND – Calreticulin ↑ ND ↓↓ 12 SERCA, PLB ↑ ANF, β-MHC ↑ Connexin40 K ↓↓↓ – Cx43 ↔ Atrial ND 5 Connexin 43 K (conditional, heart) ↓↓↓ + ND Ventricular ↔ 4

Cx, connexin; ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; PLB, phospholamban; β-MHC, β-myosin heavy chain; ND, not determined.

1224 The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 10 induced in hypertrophy are not found modify the expression of the tube formation. Genes Dev. 11:1048–1060. in the overexpressing mice. To more that are dysregulated in human dis- 10. de la Pompa, J.L., et al. 1998. Role of the NF-ATc transcription factor in morphogenesis of cardiac accurately study the consequences of ease, either experimentally or, ulti- valves and septum. Nature. 392:182–186. calreticulin re-expression in the adult mately, for therapeutic purposes. 11. Ranger, A.M., et al. 1998. The transcription factor heart, an inducible expression system NF-ATc is essential for cardiac valve formation. Nature. 392:186–190. might be developed to turn expression Acknowledgments 12. Sato, Y., et al. 1998. Cardiac-specific overexpres- on and off at later time points. A. Maass is supported by a grant from sion of mouse cardiac calsequestrin is associated The intriguing overlap in the pheno- the Deutsche Forschungsgemeinschaft with depressed cardiovascular function and hypertrophy in transgenic mice. J. Biol. Chem. types of calreticulin- or calsequestrin- (Ma 2185/1-1), and L.A. Leinwand is 273:28470–28477. overexpressing mice with mice carry- supported by NIH grant HL-50560. 13. Hunter, J.J., and Chien, K.R. 1999. Signaling path- ing targeted disruption of one of the ways for cardiac hypertrophy and failure. N. Engl. Connexin genes (summarized in Table J. Med. 341:1276–1283. 1. Michalak, M., Corbett, E.F., Mesaeli, N., Naka- 14. Houser, S.R., Piacentino, V., and Weisser, J. 2000. 1) makes a compelling case for exam- mura, K., and Opas, M. 1999. Calreticulin: one Abnormalities of calcium cycling in the hyper- ining the expression of these genes in protein, one gene, many functions. Biochem. J. trophied and failing heart. J. Mol. Cell. Cardiol. human heart disease. Postnatal devel- 344:281–292. 32:1595–1607. 2. Mesaeli, M., et al. 1999. Calreticulin is essential 15. Freeman, K., et al. 2001. Alterations in cardiac opment of cardiac conduction is for cardiac development. J. Cell Biol. 144:857–868. adrenergic signaling and calcium cycling differ- remarkably different between mice 3. Nakamura, K., et al. 2001. Complete heart block entially affect the progression of cardiomyopathy. and humans, in that increas- and sudden death in mice overexpressing calreti- J. Clin. Invest. 107:967–974. culin. J. Clin. Invest. 107:1245–1253. 16. Minamisawa, S., et al. 1999. Chronic phospho- es with age in mice, whereas it decreas- 4. Gutstein, D.E., et al. 2001. Conduction slowing lamban-sarcoplasmic reticulum calcium ATPase es with age in humans. The molecular and sudden arrhythmic death in mice with car- interaction is the critical calcium cycling defect in mechanisms underlying acquired con- diac-restricted inactivation of Connexin43. Circ. dilated cardiomyopathy. Cell. 99:313–322. Res. 88:333–339. 17. Molkentin, J., and Dorn, G., II. 2001. Cytoplasmic duction disorders (other than for 5. Hagendorff, A., Schumacher, B., Kirchhoff, S., signaling pathways that regulate cardiac hyper- ischemia-related conduction blocks) Luederitz, B., and Willecke, K. 1999. Conduction trophy. Annu. Rev. Physiol. 63:391–426. remain completely obscure. Myocar- disturbances and increased atrial vulnerability in 18. Wier, W.G., and Balke, C.W. 1999. Ca2+ release connexin40-deficient mice analyzed by trans- mechanisms, Ca2+ sparks and local control of dial biopsies taken from patients esophageal stimulation. Circulation. 99:1508–1515. excitation-contraction coupling in normal heart undergoing pacemaker implantation 6. Johnson, S., Michalak, M., Opas, M., and Eggle- muscle. Circ. Res. 85:770–777. might shed some light on gene or pro- ton, P. 2001. The ins and outs of calreticulin: 19. Orth, T., Dorner, T., Meyer zum Bueschenfelde, from the ER lumen to the extracellular space. K.H., and Mayet, W.J. 1996. Complete congenital tein expression changes if they are Trends Cell Biol. 11:122–129. heart block is associated with increased autoan- global and not restricted to cells of the 7. Rauch, F., Prud’homme, J., Arabian, A., Dedhar, tibody titers against calreticulin. Eur. J. Clin. Invest. conduction system. A recent publica- S., and St-Arnaud, R. 2000. Heart, brain, and 26:205–215. body wall defects in mice lacking calreticulin. 20. Moak, J.P., et al. 2001. Congenital heart block: tion has demonstrated the feasibility Exp. Cell Res. 256:105–111. development of late-onset cardiomyopathy, a pre- of focal gene transfer to modify elec- 8. Coppolino, M.G., et al. 1997. Calreticulin is essen- viously underappreciated sequela. J. Am. Coll. Car- trical conduction in an animal model tial for integrin-mediated and diol. 37:238–242. cell adhesion. Nature. 386:843–847. 21. Donahue, K.J., et al. 2000. Focal modification of (21). If the molecular mechanisms 9. Kuo, C.T., et al. 1997. GATA4 transcription factor electrical conduction in the heart by viral gene were known, it might be possible to is required for ventral morphogenesis and heart transfer. Nat. Med. 6:1395–1398.

The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 10 1225