Control of Cardiac Growth and Function by Calcineurin Signaling

The central role of calcium in the control of cardiac function is well established. The release and reuptake of calcium by the sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; CnA, calcineurin A subunit; CnB, calcineurin B subunit; CsA, cyclosporin A; GSK3β, glycogen synthase kinase-3 β; HDAC, histone deacetylase; MCIP1, modulatory calcineurin interacting protein 1; MEF2, myocyte enhancer factor-2; α-MHC, α-myosin heavy chain; NF-AT, nuclear factor of activated T-cells; PKG, protein kinase G; PLB, phospholamban; RyR, ryanodine receptor; SERCA2a, SR calcium-ATPase. controls not only the contraction of the cardiac myocyte but, ultimately, the heartbeat. However, the role of calcium as a key second messenger in signal transduction pathways that control growth of the heart has only recently been recognized. The calcium-dependent protein phosphatase calcineurin, also called protein phosphatase 2B, is a critical transducer of calcium signals that govern cardiac growth during development and disease. The actions of calcineurin are dependent on an array of effector proteins that influence its enzymatic activity, subcellular distribution, and stability. Additional proteins transmit calcineurin-dependent signals to the nucleus with consequent changes in gene transcription. Calcineurin signaling affects the functions of a wide range of cell types and although many of its effectors are ubiquitous, others are restricted to cardiac (and skeletal) muscle, providing muscle specificity to calcineurin signaling. The diversity of calcineurin effectors provides entry points into the signaling pathways that govern cardiac growth and function and provides opportunities for pharmacological and genetic modification of these processes. Here we discuss the functions of calcineurin and its effectors, the possibilities and pitfalls involved in its modulation as a therapeutic target in the heart, and questions for the future. Changes in calcium signaling have short and long term effects on cardiac function (1Marks A.R. J. Clin. Invest. 2003; 111: 597-600Crossref PubMed Scopus (79) Google Scholar). During cardiac contraction, or systole, calcium uptake through L-type calcium channels in the sarcolemma induces intracellular calcium release from the SR by the ryanodine receptor (RyR) (Fig. 1). Calcium is then transported back into the SR during relaxation, or diastole, through the SR calcium-ATPase (SERCA2a). The activity of SERCA2a is controlled by phospholamban (PLB), a signal-responsive inhibitor of its activity. A growing body of evidence suggests that changes in calcium handling in response to pathological (e.g. pressure overload or ischemic damage) or physiological (e.g. exercise) stimuli influences not only cardiac contractility but also cardiac growth (2Frey N. McKinsey T.A. Olson E.N. Nat. Med. 2000; 6: 1221-1227Crossref PubMed Scopus (287) Google Scholar). The adult heart grows by hypertrophy, which is mediated by an increase in myocyte size without an increase in myocyte number. Whereas hypertrophy in response to pathological stimuli is thought to be an initial salutary response to normalize ventricular wall stress and sustain cardiac output, chronic hypertrophy can progress to dilated cardiomyopathy with associated fibrosis, arrhythmias, and sudden death. Indeed, hypertrophy is a strong predictor for morbidity and mortality and is the single most important risk factor for heart failure in humans (3Benjamin E.J. Levy D. Am. J. Med. Sci. 1999; 317: 168-175Crossref PubMed Google Scholar). Whether physiological hypertrophy and pathological hypertrophy are controlled by the same or different pathways is a central issue in the field with significant clinical implications because enhancing the former and suppressing the latter could have profound therapeutic consequences. Calcineurin is a calcium/calmodulin-dependent serine/threonine protein phosphatase composed of a catalytic A subunit (CnA) and a regulatory B subunit (CnB) (reviewed in Ref. 4Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1117) Google Scholar). It can be distinguished from the more abundant protein phosphatases 1 and 2A by its sensitivity to inhibition by the immunosuppressants cyclosporin A (CsA) and FK-506, and insensitivity to okadaic acid and calyculin A. Calcineurin is also unique in its specific responsiveness to sustained, low frequency calcium signals. The activation of calcineurin occurs through the binding of calcium/calmodulin, which displaces an autoinhibitory domain of the CnA subunit (Fig. 2). The most well characterized substrate of calcineurin is the transcription factor nuclear factor of activated T-cells (NF-AT), so named because of its role in the activation of T-cells during the immune response (5Crabtree G.R. Olson E.N. Cell. 2002; 109: S67-S79Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar). Calcineurin dephosphorylates multiple serine residues near the N termini of NF-AT proteins leading to their translocation from the cytoplasm to the nucleus where they engage a variety of transcription factors and activate calcineurin-responsive genes (6Beals C.R. Clipstone N.A. Ho S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (342) Google Scholar, 7Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar, 8Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Rephosphorylation of the same sites by glycogen synthase kinase-3β (GSK3β) and other kinases promotes nuclear export of NF-AT and terminates the calcineurin signal to the nucleus. Pathological cardiac hypertrophy is coupled to the activation of a “fetal” gene program in which proteins involved in contractility, calcium handling, and energy metabolism of the fetal heart are up-regulated with consequent changes in cardiac function. Members of the GATA and myocyte enhancer factor-2 (MEF2) families of transcription factors regulate, either directly or indirectly, fetal cardiac genes before birth and in response to stress signals in the adult heart (9Passier R. Zeng H. Frey N. Naya F.J. Nicol R.L. McKinsey T.A. Overbeek P. Richardson J.A. Grant S.R. Olson E.N. J. Clin. Invest. 2000; 105: 1395-1406Crossref PubMed Scopus (422) Google Scholar, 10Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (154) Google Scholar). The link between calcium and calcineurin as a key signal transduction pathway in the heart was first recognized by the discovery that NF-ATc4, one of four NF-AT proteins, associates with GATA4, a zinc finger transcription factor (11Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2219) Google Scholar), thereby providing a potential connection between calcium-dependent signaling and fetal gene activation. The initial description of calcineurin as a mediator of cardiac hypertrophy demonstrated that overexpression of an activated CnA subunit under control of the cardiac-specific α-myosin heavy chain (α-MHC) promoter in transgenic mice led to severe cardiac hypertrophy and eventually to heart failure and sudden death (11Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2219) Google Scholar). This phenotype could be largely recapitulated by cardiac expression of a constitutively nuclear form of NF-ATc4. Subsequent studies demonstrated that inhibition of calcineurin activity with CsA and FK-506 suppressed hypertrophy in response to a variety of pathological stimuli, although conflicting results have been reported (summarized in Ref. 12Molkentin J.D. Circ. Res. 2000; 87: 731-738Crossref PubMed Scopus (199) Google Scholar). Targeted disruption of the gene encoding the CnAβ isoform has also provided support for the role of calcineurin in pathological hypertrophy (13Bueno O.F. Wilkins B.J. Tymitz K.M. Glascock B.J. Kimball T.F. Lorenz J.N. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4586-4591Crossref PubMed Scopus (220) Google Scholar). CnAβ null mice have smaller hearts than normal and show a markedly impaired hypertrophic response to infusion of angiotensin II or isoproterenol as well as to aortic constriction. The measurable activity of calcineurin is elevated in response to several hypertrophic stimuli including β-adrenergic infusion, pressure overload, and exercise (14Zou Y. Yao A. Zhu W. Kudoh S. Hiroi Y. Shimoyama M. Uozumi H. Kohmoto O. Takahashi T. Shibasaki F. Nagai R. Yazaki Y. Komuro I. Circulation. 2001; 104: 102-108Crossref PubMed Scopus (99) Google Scholar, 15Zou Y. Hiroi Y. Uozumi H. Takimoto E. Toko H. Zhu W. Kudoh S. Mizukami M. Shimoyama M. Shibasaki F. Nagai R. Yazaki Y. Komuro I. Circulation. 2001; 104: 97-101Crossref PubMed Scopus (114) Google Scholar, 16De Windt L.J. Lim H.W. Bueno O.F. Liang Q. Delling U. Braz J.C. Glascock B.J. Kimball T.F. del Monte F. Hajjar R.J. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3322-3327Crossref PubMed Scopus (179) Google Scholar). Increased calcineurin activity has also been shown to accompany hypertrophy in response to overexpression of Kv4.2N, a cardiac calcium channel (17Sah R. Oudit G.Y. Nguyen T.T. Lim H.W. Wickenden A.D. Wilson G.J. Molkentin J.D. Backx P.H. Circulation. 2002; 105: 1850-1856Crossref PubMed Scopus (57) Google Scholar). Increases in calcineurin activity often correlate with increases in calcineurin protein levels (18Lim H.W. De Windt L.J. Steinberg L. Taigen T. Witt S.A. Kimball T.R. Molkentin J.D. Circulation. 2000; 101: 2431-2437Crossref PubMed Scopus (135) Google Scholar); whether this reflects enhanced stability of the calcineurin protein or transcriptional activation of the calcineurin gene, or both, has not been determined. Sustained intracellular calcium concentrations rather than transient pulses are required to maintain nuclear localization of NF-AT (19Timmerman L.A. Clipstone N.A. Ho S.N. Northrop J.P. Crabtree G.R. Nature. 1996; 383: 837-840Crossref PubMed Scopus (472) Google Scholar). Given that intracellular calcium levels in cardiomyocytes change by 10-fold with every heartbeat, a pertinent question is how calcineurin activity might be chronically regulated in vivo. This may not occur through a global intracellular rise in calcium but perhaps through increases in calcineurin protein levels as discussed above, local changes in intracellular calcium pools, or through the actions of modulatory proteins. Despite the wealth of information linking calcineurin and the progression of cardiac hypertrophy, little is known of the exact mechanism by which calcineurin promotes growth of the cardiac myocyte, as few calcineurin substrates other than NF-AT that may contribute to the etiology of hypertrophy have been identified. Although activation of NF-AT appears to be sufficient to promote hypertrophy and NF-ATc3 null mice have a blunted hypertrophic response (20Wilkins B.J. De Windt L.J. Bueno O.F. Braz J.C. Glascock B.J. Kimball T.F. Molkentin J.D. Mol. Cell. Biol. 2002; 22: 7603-7613Crossref PubMed Scopus (218) Google Scholar), NF-AT gene targets in the cardiac myocyte also remain to be identified. NF-AT may cooperate with GATA4 in the activation of hypertrophic gene markers (10Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (154) Google Scholar, 21van Rooij E. Doevendans P.A. de Theije C.C. Babiker F.A. Molkentin J.D. de Windt L.J. J. Biol. Chem. 2002; 277: 48617-48626Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). There is also evidence from other cell types that NF-AT may directly interact with MEF2 (22Blaeser F. Ho N. Prywes R. Chatila T.A. J. Biol. Chem. 2000; 275: 197-209Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 23Youn H.D. Chatila T.A. Liu J.O. EMBO J. 2000; 19: 4323-4331Crossref PubMed Scopus (182) Google Scholar), a MADS (MCMI, agamous, deficiens, serum response factor) box transcription factor activated during cardiac hypertrophy (9Passier R. Zeng H. Frey N. Naya F.J. Nicol R.L. McKinsey T.A. Overbeek P. Richardson J.A. Grant S.R. Olson E.N. J. Clin. Invest. 2000; 105: 1395-1406Crossref PubMed Scopus (422) Google Scholar). During heart failure, signaling through the β-adrenergic pathway decreases. Normally, β-adrenergic signaling results in phosphorylation via protein kinase A of PLB, an inhibitor of SERCA2a. 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Glascock B.J. Kimball T.F. del Monte F. Hajjar R.J. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3322-3327Crossref PubMed Scopus (179) Google Scholar). Calcineurin with functions in the are discussed calcineurin interacting protein 1 was as the of the critical gene on A. I. Mol. PubMed Scopus Google Scholar). connection to calcineurin was through of the Genes Dev. 2000; Google Scholar), which functions as a of calcineurin activity. is from other protein of calcineurin because of its expression in and its by calcineurin which its expression through a of NF-AT binding sites in its gene promoter J. B. Frey N. McKinsey T.A. Olson E.N. R. Circ. Res. 2000; 87: PubMed Google Scholar). This led to the that in a inhibition to control calcineurin activity (Fig. with this overexpression of in the heart hypertrophy in response to activated calcineurin, as well as to isoproterenol infusion, aortic and B.A. McKinsey T.A. Nicol R.L. P. J. Antos C.L. R. Olson E.N. Proc. Natl. Acad. Sci. 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The exact mechanism of calcineurin myocyte is although appears to activation. overexpression of calcineurin in mice to cardiac hypertrophy, heart failure, and sudden death (11Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2219) Google Scholar). from these transgenic mice show of to sudden death D. Y. J. L. J.P. Molkentin J.D. Res. 2003; PubMed Scopus Google Scholar). are an important mechanism to the mortality and sudden death of with cardiac ventricular of hypertrophy show a phenotype of potential because of an increase in or a in R. Res. 2003; PubMed Scopus Google Scholar). The that these changes are not of transgenic mice calcineurin show a in the of channels D. Y. J. L. J.P. Molkentin J.D. Res. 2003; PubMed Scopus Google Scholar). of calcineurin activity in transgenic mice by of CsA not only hypertrophy but also a in a and transient However, because of an of the other by CsA in a of the in other was not is to the effects of CsA on A potential of linking intracellular signaling to and hypertrophy may calcineurin activity in phosphorylation of this is therapeutic to calcineurin activity may be to human The of calcineurin in cardiac including hypertrophy and failure an therapeutic or genetic inhibition of calcineurin by has a demonstrated in cardiac hypertrophy and failure in short term studies in Whether inhibition of calcineurin activity is sufficient to long term changes in the heart in response to stress signaling to be determined. The many physiological of calcineurin as in the immune also significant to its inhibition as a of or cardiac disease. in the between the calcineurin and other hypertrophic signaling as well as of effectors of calcineurin activity with cardiac entry points into the cardiac hypertrophy and heart failure and to therapeutic for the of heart disease. A. for