© 1999 American Heart Association, Inc.
Point/Counterpoint |
Signaling in Myocardial Hypertrophy
Life After Calcineurin?
From the National Heart and Lung Institute (NHLI) Division, Imperial College School of Medicine, London, United Kingdom.
Correspondence to Peter H. Sugden, DPhil, NHLI Division (Cardiac Medicine), Imperial College School of Medicine, Dovehouse St, London SW3 6LY, United Kingdom. E-mail p.sugden{at}ic.ac.uk
Key Words: myocardial hypertrophy and failure • mitogen-activated protein kinase cascade • calcineurin • calcium movement • transcription
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Introduction |
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 Top Introduction Experimental Investigation of...Before Calcineurin: Myocardial...An Involvement of Calcineurin...Do Derangements in Excitation...Mitochondrial Permeability...General Comments and Future...References |
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Cardiac (ventricular) hypertrophy is an important adaptive response in vivo that (at least in the shorter term) allows the organism to maintain or increase its cardiac output. Global ventricular hypertrophy is a recognized response to increased pressure or volume work (reviewed in Reference 11 ), with increased myofibrillogenesis and sarcomere deposition being cardinal features. Although global hypertrophy is clinically important, probably the most significant form of cardiac hypertrophy in terms of patient numbers is the localized hypertrophy of the ventricular wall that may follow loss of myocardium after a survivable myocardial infarct. In the early stages, both global and localized hypertrophy may resemble the readily reversible, nonfibrotic "physiological" hypertrophy that develops after repeated endurance exercise. In the longer term, beneficial, "compensated" hypertrophy may decay into maladaptive "decompensated" hypertrophy and heart failure (reviewed in References 2 and 32 3 ) with diminished coronary flow reserve and increased risk of lethal arrhythmias. Although some aspects of the maladapted state are probably intrinsic to the myocyte [eg, prolongation of the action potential and Ca2+ transient (reviewed in References 4 and 54 5 )], other factors (increased fibrosis and mismatch between O2 supply and demand) are also probably involved in decompensation.
The predominating view is that mammalian ventricular myocytes lose their capacity for cell division during the perinatal period and are thus terminally differentiated cells, although this is still a matter of some dispute (reviewed in References 6 and 76 7 ). In contrast, other cells in the heart (endothelial cells, fibroblasts, and smooth muscle cells) retain their mitotic capacity. Although ventricular myocytes are not the only cell type involved in the overall hypertrophic response, much of the ventricular enlargement or remodeling is attributable to their hypertrophy. The identities of signaling pathways that couple the demand for increased contractile power to increased myocyte growth and altered gene expression have been actively investigated for many years. Protein phosphorylation (catalyzed principally by Ser-/Thr- or Tyr-specific protein kinases) and phosphoprotein dephosphorylation (catalyzed by phosphoprotein phosphatases) play central roles in the regulation of many cellular events, including growth and cell division, and it is widely believed that these processes participate in myocyte hypertrophy.
From the broad point of view, 2 hypotheses of hypertrophy
have been proposed, which are the extrinsic and the intrinsic
hypotheses. The former maintains that myocyte hypertrophy
results from extracellular factors (Table 1
) that are either of a
neuroendocrine origin (eg, the catecholamines) or are
synthesized and released locally by the myocytes and
nonmyocytes in the heart (eg, endothelin-1 [ET-1] and
angiotensin II [Ang II]). The intrinsic hypothesis
maintains that changes originating within the myocyte are responsible
for hypertrophy. Because of its well-established role in
excitation-contraction coupling, increased
[Ca2+]i concentration has
been suggested to be an intrinsic mediator. The 2 hypotheses are not
mutually exclusive because, in the hands of some workers, extrinsic
agonists such as ET-1 increase
[Ca2+]i concentrations in
myocytes (reviewed in References 8 and 98 9 ) and, conversely,
[Ca2+]i is known
to be involved in the secretion of peptide hormones (eg, secretion of
atrial natriuretic factor [ANF] from
myocytes10 ). The problem with the
Ca2+ hypothesis is that, although
[Ca2+]i has been
implicated in the hypertrophic response,11 12 no clearly
defined coupling mechanism has been substantiated until recently. As
discussed in the accompanying article,13 the recent
elegant studies of Molkentin et al14 have provided
evidence that the Ca2+/calmodulin
(CaM)–dependent phosphoprotein phosphatase calcineurin (also known as
protein phosphatase 2B) plays a significant role in myocyte
hypertrophy. The excitement generated by this finding was
demonstrated by the rapid appearance of editorials in
Science15 and Nature
Medicine.16 The problem is that there will be an
inevitable temptation to believe that the biochemical mechanisms
underlying myocyte hypertrophy and heart failure have been
elucidated in all but detail and that therapeutic intervention is now
readily at hand. Indeed, in a review of a recent manuscript that we
submitted, one reviewer made the following comment: "The authors talk
a lot about the mitogen-activated protein kinase (MAPK) pathway
and hypertrophy and omit any mention of the recent and
exciting observations about the role of Ca2+ and
calcineurin-dependent activation of NFAT [nuclear factor of
activated T lymphocytes] transcription factors in
hypertrophy. At this point, it seems likely that many of
the hypertrophic effects of the agonists used here are mediated by the
calcineurin–NFAT pathway rather than the MAPK pathway." In my
opinion, this comment crystallizes the dangers that Molkentin et
al14 have unintentionally created, and I am convinced that
the issue is far from settled. There are 3 main points that I wish to
cover. First, is there evidence for the involvement of other (possibly
interacting) signaling pathways leading to hypertrophy?
Second, how convincing is the evidence for involvement of calcineurin?
Third, if calcineurin is involved, how convincing is the mechanism
proposed by Molkentin et al,14 namely
dephosphorylation of the transcription factor nuclear
factor of activated T-lymphocytes-3 (NFAT3)?
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Experimental Investigation of Myocardial Hypertrophy |
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Hypertrophy in Vitro
The most commonly used in vitro model involves primary culture of neonatal rat ventricular myocytes, and this has allowed the biochemical and molecular characteristics of hypertrophy to be studied in detail under controlled conditions (reviewed in References 17 through 1917 18 19 ). A plethora of hypertrophic factors and interventions has been identified (Table 1
). It is invidious to
attempt to identify a single hypertrophic agonist, and many may
participate in vivo. Although there is concern about the fidelity of
simulation of the adult hypertrophic response, many of the changes seen
in the cultured neonatal cells have also been detected in adult animals
in vivo (reviewed in Reference 1717 ). When exposed to hypertrophic
stimuli, myocytes in culture show increased myofibrillogenesis, and the
cells enlarge. From a biological standpoint, accumulation of sarcomeres
is perhaps the most important feature of cardiac
hypertrophy, because, above all other manifestations, this
allows the myocyte to increase its contractile power. In addition,
there are alterations (both upregulation and downregulation) in gene
expression. Immediate-early genes that encode transcription factors
(c-fos, c-jun, and Egr-1) are rapidly
(within 1 hour) and transiently upregulated, and these may be important
in the regulation of genes encoding myofibrillar proteins. B-type
natriuretic peptide (BNP) is expressed at this early stage,
and this is maintained.20 In the medium term (12 to
24 hours), the "fetal" pattern of gene expression is recapitulated
with reexpression of ANF, skeletal muscle 
-actin, and ß-myosin
heavy chain (ß-MHC). Although now being superseded by BNP,
reexpression of ANF has been widely used as an index of
hypertrophy. Over 24 to 48 hours, other genes are
upregulated (ventricular myosin light chain 2, cardiac
muscle -actin). Expression of some genes is downregulated (eg,
sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2
[SERCA2], reviewed in Reference 2121 ), whereas the expression of others
is relatively unchanged. Gene expression is regulated by the binding of trans-acting transcription factors and other cooperating proteins (eg, RNA polymerases) to cis-acting consensus recognition sequences that lie upstream (5') from the transcriptional initiation sites in genes (the promoter region) or to more distant enhancer regions. For most genes, these regions contain multiple sequences recognized by a variety of transcription factors (for a review on the heart, see Reference 2222 ), and interactions between the various DNA-bound transcription factors occur. Gene expression is most commonly studied by transient transfection of expression plasmids containing fusion genes of the 5'-upstream regulatory sequences of hypertrophic "marker" genes (eg, ANF) coupled to suitable reporter genes. This has allowed the identification of regulatory elements within the promoter regions. By cotransfecting/coinfecting suitable reporters with plasmids or adenoviral vectors encoding signaling proteins, this approach has been used to identify putative participants in the induction of hypertrophy.
One frequently ignored fact is that hypertrophy requires increased accumulation of protein (particularly of myofibrillar proteins) and increased myofibrillogenesis. Control at the level of protein turnover and sarcomeric assembly is necessary. As reviewed in Reference 1919 , hypertrophic agonists generally increase the rate of protein synthesis in the short term by increasing the peptide chain initiation (the rate-controlling step). In the longer term, ribosomal synthesis also increases. Because of the experimental difficulties in measuring protein degradation (particularly of specific proteins) with accuracy, it is not clear whether this process is also regulated. In addition, the regulation of myofibrillogenesis is also poorly understood.
Hypertrophy In Vivo
In vivo, probably the most commonly used surgical intervention
involves coarctation of the aorta, which induces a pressure-overload
hypertrophy. It is important to note that although the
aorta can be coarcted at a variety of levels to induce pressure
overload, the models may not be necessarily equivalent.23
A variety of transgenic mouse models of cardiac hypertrophy
or failure have been developed (reviewed in References 24 through 2624 25 26 ).
Although transgenic "knockouts" have been used, most studies have
involved expression of a wild-type transgene or one encoding a
constitutively active form of a protein. The transgene is
cardiospecifically expressed by placing it under the regulation of a
constitutive cardiomyocyte promoter (often the -MHC
promoter sequence). One problem is that transgene expression is
difficult to control. In the mouse, mRNA for both -MHC and ß-MHC
is expressed from the onset of cardiogenesis, with ß-MHC mRNA
predominating during fetal development.27 This is reversed
during the perinatal period, with a switch occurring in relative
abundances of transcripts (-MHC mRNA:ß-MHC mRNA, 
16:1). This
pattern is maintained during adult life in control mice. There are
several points here. First, because of the leakiness of the -MHC
promoter, placing a transgene under its control may still allow
transgene expression at critical stages in cardiac development. Second,
the recapitulation of ß-MHC expression and the decline in -MHC
expression during hypertrophy should lead to
down-regulation of transgene expression at this stage. Third,
transgenesis may modify expression of other genes through obscure
mechanisms (see, eg, References 28 and 2928 29 ). Fourth, there may be
considerable variability in phenotype and penetrance between
individuals expressing any single transgene.30 Fifth, the
phenotype is often influenced by the strain of mouse used. In
the future, it is likely that use of inducible systems will allow more
highly regulated expression of heterologous transgenes, but, even here,
transgene expression may be difficult to control uniformly.
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Before Calcineurin: Myocardial Hypertrophy and Signaling Through Protein Kinase C (PKC) and MAPK Cascades |
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Despite differences in emphasis, there has been relatively general agreement that activation of PKC and 1 or more of the MAPK cascades (Figure 1
) by G protein–coupled receptor
(GPCR) agonists induces many of the features of hypertrophy
(see also Table 2). Since we have
reviewed this topic in depth recently,19 31 32 only the
salient features will be outlined. I do not wish to exclude involvement
of other protein kinases (eg, the Janus kinases,33 34 35
Ca2+/CaM kinase II,36 and most
recently myosin light chain kinase,37 ), but work on these
has been less extensive. MAPK cascades consist of 3-member protein
kinase "modules" (Figure 1). The MAPK kinase kinases (MKKKs)
are Ser-/Thr-specific kinases that phosphorylate and
activate MAPK kinases (MKKs), and the MKKs in turn
phosphorylate and activate the MAPKs, the cascade
thus allowing signal integration and amplification. MAPKs are unusual
in that they require phosphorylation of a Tyr and a Thr
residue in a Thr-Xaa-Tyr motif that lies on their surface in a
regulatory loop of varying length, and the sequence of this tripeptide
represents 1 criterion for MAPK classification. After their
activation, MAPKs phosphorylate a broad range of substrates
(including nuclear transcription factors, other protein kinases, and
various signaling molecules), thereby modifying their biological
activities. Although MAPKs preferentially phosphorylate
(Ser/Thr)–Pro sequences (or, even better,
Pro-Xaa-[Ser/Thr]–Pro38 ; reviewed in Reference 3939 ),
this dipeptidyl sequence is relatively common in proteins, and other
factors also contribute to specificity (reviewed in Reference 4040 ).
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GTP-binding proteins (G proteins) participate in the activation of MAPK cascades, and these can be broadly divided into 2 types: the heterotrimeric (GPCR-associated) group and the p21 small G protein superfamily, of which there are 5 subgroups (prototypically Ras, but also Rho, ADP ribosylation factors, Rab, and Ran). Of the heterotrimeric G proteins, the Gq/G11 subfamily (Gq for simplicity) has been shown most convincingly to be associated with hypertrophy. From the p21 small G protein superfamily, only the Ras and Rho subgroups (reviewed in References 41 and 4241 42 , respectively) have been examined from this standpoint. All G proteins are biologically inactive in their GDP-ligated form and become activated on exchange of GDP for GTP. Their innate GTPase activity returns them to the inactive state (the GDP/GTP cycle), but this process can be stimulated by ancillary proteins. A number of proteins also affect GDP/GTP exchange (the GPCRs in the case of heterotrimeric G proteins, stimulatory guanine nucleotide exchange factors such as Sos, and inhibitory factors for the small G proteins).
I will use ET-1 as my principal example of a hypertrophic GPCR agonist,
as there is unanimity that it is strongly hypertrophic. By binding to
heptahelical transmembrane GPCRs, agonists such as ET-1 stimulate the
dissociation of membrane-bound inactive heterotrimeric
Gq proteins [(q
· GDP) · ß
] into q · GTP
and ß dimers. Both q · GTP and
ß dimers are potentially capable of activating phospholipase Cß
isoforms leading to the hydrolysis of membrane phosphatidylinositol
4,5-bisphosphate (PtdInsP2) to inositol
1,4,5-trisphosphate (InsP3) and diacylglycerols
(DGs). DGs are retained in the plane of the membrane, and the
DG-sensitive isoforms of PKC (principally nPKC
and nPKC
in the
rat ventricular myocyte43 44 ) translocate to
the particulate fraction (and presumably become activated).
Phorbol esters such as phorbol 12-myristate 13-acetate (PMA)
act as DG analogs and also translocate myocytic nPKC and
nPKC,45 46 and they are also strongly
hypertrophic.47 48 The next step(s) in the signaling
pathway is unclear, but the end result is that both ET-1 and PMA
activate membrane-bound Ras (Ras · GDP
Ras ·
GTP) (A. Chiloeches, P.H. Sugden, unpublished data, 1998).
One of the best-defined roles of Ras · GTP is its activation of
the extracellular signal–regulated protein kinase (ERK) cascade
(Figure 1), which is particularly involved in cell growth,
division, and differentiation. The ERK cascade MKKKs are members of the
Raf family (c-Raf, A-Raf, and B-Raf), the MKKs are MKK1 and MKK2, and
the MAPKs (phosphorylation motif Tyr-Glu-Thr) are ERK1
(p44-MAPK) and ERK2 (p42-MAPK). ET-1 and PMA strongly activate
all 3 stages of the ERK cascade (Raf,49 MKK1 and
-2,50 and ERKs43 50 51 ) in myocytes.
Activation of Raf involves its binding to membrane-localized Ras
· GTP and its translocation to this fraction. It is not clear whether
this interaction is sufficient to activate Raf or whether other
events such as Ser/Thr phosphorylation and/or Tyr
phosphorylation (possibly involving nonreceptor protein
Tyr kinases of the Src family52 53 ) are additionally
required. The potential for ERKs to regulate gene transcription in the
myocyte is demonstrated by the rapid appearance (within 4 minutes) of
active phospho-ERKs in nuclei of myocytes exposed to PMA (H.F.
Paterson, P.H. Sugden, unpublished data, 1998).
Depending on the criteria used, there are an additional 2 (see, eg,
Reference 3232 ) or 5 (see, eg, Reference 5454 ) subgroups of the MAPK
superfamily. The simpler classification based on the sequences of the
tripeptide phosphorylation motif will be used here. The
c-Jun N-terminal kinases (JNKs; phosphorylation motif
Thr-Pro-Tyr) and the p38-MAPKs (phosphorylation motif
Thr-Gly-Tyr) were first identified as being activated by
cellular stresses (eg, protein synthesis inhibitors,
hyperosmotic shock, and reactive oxygen species), and this is certainly
true in myocytes.55 56 57 It has also emerged that
Gq protein–coupled receptor
(GqPCR) agonists such as ET-1 are moderately
effective in activating JNKs55 and p38-MAPK57
in these cells but that PMA is a poor
activator.55 57 The upstream signaling
pathways are not as clear as in the ERK cascade, although MKKs, MKKKs,
other protein kinases, and small G proteins (particularly Rac and
Cdc42, 2 members of the Rho subfamily) are likely to be involved. As
with the ERKs, activation of JNKs and p38-MAPKs leads to
phosphorylation of transcription factors (Figure 1) and other signaling molecules. Generally speaking, the
biological roles of the JNKs and p38-MAPKs are less clear than those of
the ERKs. In addition to their hypertrophic actions in myocytes (Table 2), activation of JNKs has been associated with
apoptosis in nonmyocytes and
myocytes,58 59 whereas the p38-MAPKs have been associated
with both apoptosis60 and cytoprotection/cell
survival57 59 61 in myocytes (and other cells).
There is considerable evidence that activation of any of the 3 MAPK
cascades can lead to a hypertrophic response in myocytes. All 3 MAPK
subfamilies are activated by ET-1 and other hypertrophic GPCR
agonists,43 50 51 55 57 and the novel hypertrophic agonist
prostaglandin F262 63 has
recently been shown to activate JNKs.64
Transfection/infection of suitably constitutively activated
components of the cascades (Table 2) leads to induction of many
features of the hypertrophic response, although myofibrillogenesis is
not always observed. Transfection/infection of "dominant-negative"
(inhibitory) mutant components of the cascades opposes the
hypertrophic actions of GPCR agonists, as does downregulation of ERKs
by antisense oligodeoxynucleotides65 or
transfection of phospho-MAPK phosphatases.66 67 It is
evident from Table 2 that there is still considerable confusion
in this area, and the reasons for the variations between experimental
groups are unclear (reviewed in Reference 3232 ). Two points should be
borne in mind. First, the degree of overexpression may mean that the
experiments have little relation to the
physiological situation. Second, the regulation of
transcription from genomic DNA may be quite different from that from
episomal (plasmid) DNA because of the influence of chromatin proteins.
In the hands of some (but not all68 69 ) investigators,
selective inhibitors of the ERK and p38-MAPK cascades
(PD98059 for the ERK cascade57 and SB203580 or SB202190
for the p38-MAPK cascade60 61 70 ) diminish the
hypertrophic response to GPCR agonists, although the interpretation of
these studies may not be simple.57 The overall conclusion
is that MAPK cascades participate in the hypertrophic response. The
ensuing steps are unclear, but it is presumed that transcription factor
phosphorylation and transcriptional activation follow,
and ET-1 certainly induces phosphorylation and
upregulation of c-Jun protein in myocytes.71 Furthermore,
the interaction of the novel transcription factor ATF6 with the
ANF promoter region, which may be responsible for inducible expression
of ANF, may be stimulated by p38-MAPK–dependent
phosphorylation of ATF6.72 Despite some
anomalies,66 68 73 I personally still favor a
predominating role for the ERKs, and I regard the hypertrophic response
as an attempt at "transformation" in a terminally differentiated
cell.
In addition, activation of participating signaling molecules proximal
to the MAPK cascades leads to a hypertrophic phenotype. With
particular reference to the ERK cascade, it is clear that transient
transfection of myocytes with constitutively activated forms of
the Gq subunit,69 74 PKC
isoforms,75 76 77 Ras,78 or
Src79 80 leads to a hypertrophic response. Inhibition of
Gq signaling in transgenic mice by disruption
of coupling between the GqPCR and
Gq protein diminishes the hypertrophy
associated with pressure overload and diminishes the activation of ERKs
by GqPCR agonists.81 Except for Src
(which has not been tested), cardiospecific overexpression of each of
these signaling molecules leads to the development of cardiac
hypertrophy and heart failure in transgenic mouse
models,30 82 83 84 85 86 although some investigators have
interpreted their results as indicating that the
hypertrophy is independent of ERKs (see, eg, Reference 8484 ).
One problem with this interpretation is that ERK was only examined at
the heart failure end stage. Activation of ERKs by hypertrophic
agonists is transient,50 and they probably trigger
responses. Prolonged activation would therefore not be anticipated.
Participation of the Rho family of small G proteins (Rho isoforms
themselves [eg, RhoA], Rac, and Cdc42, reviewed in Reference 4242 ) in
hypertrophy has also been examined. In a variety of
nonmyocytic cell lines, the Rho family regulates cell morphology and
actin stress fiber assembly. In addition, Rac and Cdc42 appear to be
involved in activation of the JNK and p38-MAPK cascades, but Rho
isoforms are not thought to activate these cascades. Although
less effective than constitutively activated Ras,
constitutively activated Rac is hypertrophic.70
Signaling through Rho itself is poorly understood. Two groups of
Rho-activated protein kinases (Rho kinase/ROK and PRK/PKN
groups) have been identified (reviewed in Reference 8787 ), the PRK/PKN
group being related to PKC (reviewed in Reference 8888 ). Recent evidence
suggests that, in addition to Ras, hypertrophy induced by
or mediated through Gq may also require
signaling through RhoA69 89 90 91 92 and that Rho kinase/ROK
may mediate this effect.92 However, it is not clear
whether hypertrophic agonists activate Rho or the various
Rho-dependent kinases.
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An Involvement of Calcineurin in Myocardial Hypertrophy and Heart Failure? |
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Calcineurin (reviewed in References 93 and 9493 94 ) is a ubiquitously expressed phosphatase that is activated by micromolar Ca2+ concentrations. It is a heterodimer of a 60- to 65-kDa calcineurin A subunit and a 19-kDa calcineurin B subunit. All calcineurin A isoforms contain N-terminal and C-terminal variable domains, a catalytic domain, and a regulatory domain. The regulatory domain contains a calcineurin B-binding helix, a CaM-binding domain, and an autoinhibitory region that binds to the active site preventing enzymic activity at low Ca2+ concentrations. A constitutively activated mutant calcineurin A (calcineurin A[1
398]) has been described in which about two thirds
of the N-terminal region of the regulatory domain has been
deleted.95 The deleted region includes most of the
CaM-binding domain, the autoinhibitory domain, and the
C-terminal variable region, but the calcineurin B and immunophilin
(see below) binding sites are intact. Increasing the
Ca2+ concentration into the micromolar range
causes a small activation in the absence of CaM, but in the presence of
CaM, there is a highly cooperative activation of calcineurin. The role
of calcineurin B in the regulation of enzymic activity by
Ca2+ is obscure. It is essential for enzymic
activity (perhaps more so than CaM), but the suggestion is that it
fulfils a structural role.
One exciting function of calcineurin is its involvement in
NFAT-mediated T-lymphocyte activation (reviewed in References 94, 96,
and 9794 96 97 ). NFATs (reviewed in References 94 and 9794 97 ) are a family of Rel
homology transcription factors, a group that also includes nuclear
factor 
B. In the inactive state, NFATs are
phosphorylated and (like nuclear factor B) are
retained in the cytoplasm. NFATs are tightly associated with
calcineurin, and this association is apparently independent of the
phosphorylation state of NFAT. When T lymphocytes bind
to antigen-presenting cells through T-lymphocyte receptors,
cytoplasmic Ca2+ concentrations increase and
activate calcineurin, leading to NFAT
dephosphorylation (Figure 2). This allows NFAT to migrate into the
nucleus (still complexed to calcineurin98 ), where it
upregulates the transcription of a number of genes, including those for
interleukin-2 (an autocrine growth factor in T lymphocytes) and other
lymphocyte growth factors. The increase in cytoplasmic
Ca2+ is mediated by phospholipase C–catalyzed
hydrolysis of PtdInsP2 to
InsP3, with concomitant release of
Ca2+ from intracellular stores. In addition,
muscarinic GqPCR-mediated activation of NFATs has
been described in lymphocytes and other cells.99 100 It is
not clear which protein kinases maintain NFAT in its inactive state in
the cytoplasm in any cell type, and this remains an important question.
Candidates include JNKs,101 casein kinase I in
conjunction with MAPK/ERK kinase kinase (MEKK1, an
MKKK),102 and glycogen synthase kinase
3.103 104 Calcineurin is the target of the
immunosuppressants cyclosporin A (CsA) and FK-506 in T lymphocytes
(reviewed in References 94 and 9794 97 ). These bind to their respective
binding proteins, the immunophilins cyclophilin A and 12- and 12.6-kDa
FK-506 binding proteins (FKBP12 and FKBP12.6, respectively). The
CsA-cyclophilin and FK506-FKBP12 complexes bind to the calcineurin
heterodimer and prevent its activation. In T cells, CsA inhibits the
activation of the JNK and p38-MAPK cascades by an unknown
mechanism.105 This observation may be relevant to
hypertrophy, in which (as described above) both of these
cascades have been implicated.
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Calcineurin and Cardiac Hypertrophy/Heart Failure
The transcription factor GATA4106 (reviewed in
Reference 107107 ) is present in adult heart (and a limited number of
other tissues) and is involved in the positive regulation of a variety
of hypertrophic marker genes, including ANF, BNP, and
ß-MHC.108 109 GATA4 interacts with other transcription
factors, and recent work has suggested that ANF expression may be
regulated by cooperative interaction of cis elements with
GATA4 and Csx/Nkx2.5.110 Csx/Nkx2.5 is a homeodomain
transcription factor that is cardiospecifically expressed into
adulthood.111 112
The recent findings of Molkentin et al14 (summarized in
Reference 1313 ) implicating a
[Ca2+]icalcineurinNFAT3
pathway in cardiac hypertrophy that is analogous to T-cell
activation have caused a great deal of excitement. Using a yeast
2-hybrid screen and a mouse whole-embryo cDNA library, numerous clones
encoding proteins interacting with GATA4 were isolated. The only clone
of which the identity was reported encoded NFAT3, and details of the
identities of the other proteins are eagerly awaited. However, given
that a whole embryo library was used, these species may not be relevant
to the heart. Myocytes transiently transfected with plasmids encoding
calcineurin A(1398), NFAT3, and GATA4 dramatically increased
expression of a BNP reporter. Transfection of any 1 plasmid, or of any
2 plasmids, was much less effective. It is somewhat surprising that
calcineurin A(1398) alone did not stimulate the BNP reporter, and
this might suggest that NFAT3 and/or GATA4 are limiting. The expression
of ANF and cardiac troponin I genes was examined, but no quantitative
details were presented. It is not clear whether myofibrillar
assembly was increased in the transfected myocytes. CsA or FK-506
reduced the increases in cell size and myofibrillar assembly of
myocytes exposed to Ang II or phenylephrine for 72 hours,
as well as the Ang II–induced increase in ANF mRNA expression. This
result appears to differ from that of Boluyt et al,113 who
showed that FK-506 was ineffective in reducing
phenylephrine-induced increases in myocyte size.
Molkentin et al14 suggested that Ang II and
phenylephrine (and, inferentially, ET-1) increase
[Ca2+]i concentrations,
and indeed some workers have found this to be the case. This is by no
means universally accepted, especially in adult cardiac
myocytes.114 115 116 However, there is good evidence that
these agonists induce alkalosis in myocytes (reviewed in References 9,
117, and 1189 117 118 ) probably by activating the sarcolemmal
Na+/H+ exchanger (reviewed
in Reference 119119 ), a process known to be regulated by PKCs and MAPKs.
Given that Ca2+ binding to regulatory
"EF hands" proteins such as CaM and troponin C is mediated
by deprotonated acidic amino acids,120 121 alkalinization
will favor increased Ca2+ binding to its effector
proteins even in the absence of detectable increases in free
[Ca2+]i
concentrations.122 Thus, hypertrophic agonists could
activate calcineurin by raising
[Ca2+]i concentrations,
pHi, or both (Figure 2). This is
rather a satisfying hypothesis from another point of view, because
small increases in pHi potently stimulate cardiac
protein synthesis,123 another cardinal feature of the
hypertrophic response. Myocardial hypertrophy could be
mediated through changes in the concentrations of simple ions
(Ca2+ and H+).
Transgenic Mice
Cardiomyocytic (-MHC promoter–controlled) expression of
calcineurin A(1398) in a number of founder lines induced a cardiac
enlargement viewed by the authors as corresponding principally to
"concentric" hypertrophy,14 the type often
found in association with pressure overload. Somewhat surprisingly,
given that the active phosphatase was targeted to myocytes, increased
cardiac fibrosis was also detected. This implies that alterations
within myocytes might induce remodeling and synthesis of the
extracellular matrix, presumably by communicating with fibroblasts and
smooth muscle cells. Details of the contractile properties of the
transgenic hearts and hemodynamic measurements were not
presented, nor were details of Ca2+
transients in single myocytes. In lymphocytes, activation of
calcineurin and translocation of NFAT is favored both by sustained
plateaux of [Ca2+]i124 and by
more rapid (1 per 1 minute)
[Ca2+]i
oscillations.125 This is quite different from
the situation in the mouse heart, in which the rate of beating is
400 per 1 minute. It is not clear whether such rapid transients lead
to activation of calcineurin, and, unfortunately, it is not feasible to
measure calcineurin activity in situ. Nor is it clear to what extent
the myocardial hypertrophy and failure in these transgenic
animals represents an accurate analog of the human disease,
given the possible disturbances in development that could occur
because of "leaky" -MHC promoter activation (see above) with
activation of calcineurin during embryogenesis and fetal life.
Furthermore, it is not known whether pure myocyte
hypertrophy alone contributed to the cardiac enlargement,
or whether there was coexisting myocyte hyperplasia, as seen in
transgenic mice that overexpress CaM developmentally.126
It is interesting that myocytes from calcineurin A(1398) transgenic
animals showed alterations in nuclear morphology,14
suggesting that the protein may interfere with the cell cycle.
Cardiospecific expression of constitutively activated NFAT3
(NFAT3
317, in which the N-terminal third of the molecule
encompassing the sites of phosphorylation and
calcineurin interaction is deleted) caused hypertrophy in
transgenic mice.14 In cultured myocytes, NFAT3317
protein was localized to the nucleus, whereas the full-length protein
remained in the cytoplasm. To my mind, this represents some of
the strongest evidence that the calcineurin/NFAT3 pathway induces
myocardial hypertrophy. However, it is not clear whether
hearts from the NFAT3317 transgenic mice were similar to those from
calcineurin A(1398) transgenic animals, nor whether transfection of
NFAT3317 induced a hypertrophic phenotype in cultured
myocytes. Again, the possible involvement of NFAT in cardiac
development127 should be borne in mind.
Interaction of Calcineurin, Ca2+, and MAPK
Signaling Pathways
There is clear evidence that the calcineurin/NFAT pathway
interacts with other signaling pathways. In the nucleus, NFATs interact
with the activator protein-1 (AP-1) transcription factor
complex (c-Fos/c-Jun heterodimers) to bind to the 5'-regulatory region
of the interleukin-2 gene (reviewed in References 96 and 12896 128 ), and the
crystal structure of this complex has recently been
determined.129 Since AP-1 transactivating activity is
under the control of signals emerging from the Ras superfamily and the
MAPK cascades (eg, through phosphorylation of c-Jun,
reviewed in References 130 and 131130 131 ), these signaling pathways cooperate
with the calcineurin pathway (reviewed in References 128 and 132128 132 ).
Although currently there is no experimental evidence to suggest the
existence of regulatory interaction, the promoter region of the ANF
gene contains 2 AP-1–related cAMP responsive element (CRE)-like
consensus sequences (TGAGCTCA, at –555 to –549 bp and at –603 to
–596 bp on the sense strand) in close apposition to 2 NFAT sequences
(consensus [A/T]GGAAA, at –541 to –536 bp on the sense strand and
at –612 to –607 bp on the antisense strand). The CRE-like sequence
binds c-Jun/ATF2 heterodimers, and (as with c-Jun) ATF2 transactivating
activity is increased by MAPK-catalyzed
phosphorylation.133 134 135 The interaction
of calcineurin signaling pathways with MAPK pathways in
hypertrophy is thus a distinct possibility (Figure 2). In addition, there are already examples of interaction of
[Ca2+]i with the ERK and
JNK cascades in myocytes,12 136 137 and a recent article
has even suggested that the PKCRasRafMKK1/-2ERK pathway may
induce changes in Ca2+ transients similar to
those seen in myocardial hypertrophy and heart
failure.138 The mechanisms of these interactions remain
relatively ill defined.
Might CsA and FK-506 Be Used to Treat Cardiac
Hypertrophy and Heart Failure?
CsA has been reported to diminish the hypertrophy or
dilated cardiomyopathy seen in 3 transgenic models
involving mutations in myofibrillar proteins.139 In
addition, prophylactic administration of CsA inhibited the
development of cardiac hypertrophy after suprarenal banding
of the abdominal aorta in the rat.139 In contrast, CsA
does not prevent the cardiac hypertrophy induced by
cardiospecific expression of an activated retinoic acid
receptor,139 implying that some forms of myocardial
hypertrophy may be mediated by calcineurin-independent
signaling pathways. However, 2 separate groups have subsequently been
unable to detect any inhibitory effect of CsA or FK-506 on
pressure-overload hypertrophy induced by constriction of
the abdominal aorta in the rat140 or the transverse aorta
in the mouse.141 Although these differences may be
attributable to variations in experimental technique or to the time
course of hypertrophy in pressure-overload
models,142 they do indicate that the involvement of
calcineurin in hypertrophy in vivo has not been
unambiguously established. More importantly, from the point of view of
therapy, it is not clear whether administration of CsA during the
"decompensated" phase of hypertrophy/heart failure
(rather than prophylactic administration) is
beneficial.
CsA is a relatively toxic drug that is used clinically to counteract rejection after transplantation. In addition to problems of immunosuppression (increased opportunistic infection and skin malignancies), the drug is nephrotoxic, and this may be caused by its interference with the calcineurin-dependent regulation of convoluted tubule Na+/K+-ATPase.143 Its use is also associated with hypertension (possibly because of the nephrotoxic side effects involving renal vasoconstriction144 ), neural effects (tremor and fits), hepatotoxicity, and gingival hypertrophy/hyperplasia. A number of studies in transplant patients have examined the association of cardiac hypertrophy with the use of CsA. Although some studies have failed to detect a convincing association (eg, Reference 145145 ), others have shown that CsA may actually induce cardiac hypertrophy through its hypertensive side effects.146 147 In addition, at least 1 study has found that CsA has hypertrophic effects that are independent of its hypertensive effects.148 Although FK-506 may cause fewer side effects,144 it is more expensive than CsA. There is thus relatively little evidence that administration of immunosuppressants to human beings is antihypertrophic, and, in my view, it would be ill advised to administer them prophylactically.
Calcineurin and Specification of Skeletal Muscle Fiber
Type
Transfection of calcineurin A(1398) into C2C12 myotubes
increases transcription of slow fiber type-specific genes through NFATs
and interacting transcription factors, and administration of CsA in
intact rats increases the proportion of fast muscle fibers in the
slow-twitch postural soleus muscle.149 Characterization of
muscle fiber composition in a suitable calcineurin A(1398)
transgenic mouse is eagerly awaited. At first sight, the studies
might seem irrelevant to myocardial disease. However, in a canine model
of chronic heart failure, the proportion of slow-twitch fibers is
decreased.150 This could not be attributed to changes in
capillary density or muscle fiber destruction. Similar changes in fiber
type (combined with exercise intolerance) are seen in human heart
failure, although here there may also be fiber loss and changes in
capillary density (reviewed in Reference 151151 ). A therapeutic regime
that increases calcineurin activity and the content of slow-twitch
fiber type in skeletal muscle might therefore be beneficial in heart
failure patients. However, if calcineurin is involved in cardiac
hypertrophy, such a regimen could be detrimental to the
heart.
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Do Derangements in Excitation-Contraction Coupling Contribute to Cardiac Hypertrophy? |
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[Ca2+]i uptake and release are of major importance in the regulation of excitation-contraction coupling in the cardiac myocyte (reviewed in Reference 152152 ), and (as originally shown by Gwathmey et al153 154 ) altered Ca2+ "handling" is frequently encountered in hypertrophy and heart failure (reviewed in References 4 and 54 5 ). In the resting state, [Ca2+]i is sequestered in the sarcoplasmic reticulum (SR) by the Ca2+ binding protein, calsequestrin. After excitation, influx of small amounts of [Ca2+]o through sarcolemmal L-type Ca2+ channels activates SR Ca2+ release through the tetrameric SR Ca2+ release channel or ryanodine receptor (RyR), a process known as Ca2+-induced Ca2+-release (CICR). The type 2 RyR predominates in cardiac muscle (where CICR is very important), and the type 1 RyR predominates in skeletal muscle (where CICR is less important). Reuptake of [Ca2+]i into the SR involves SERCA2, whereas the sarcolemmal Na+/Ca2+ exchanger and sarcolemmal Ca2+ pumps are involved in the removal of [Ca2+]i from the myocyte. Could the hypertrophy/failure associated with activation of calcineurin be a manifestation of a generalized perturbation in excitation-contraction coupling that the heart interprets as representing a contractile deficit?
A number of transgenic mouse lines in which proteins that participate in [Ca2+]i handling are overexpressed or "knocked out" have been developed. CaM plays a role in the regulation of cell growth and division in eukaryotic calls (reviewed in Reference 155155 ). Developmental overexpression of CaM induces myocyte hypertrophy and hyperplasia,126 with calcineurin and CaM kinase II suggested as possible targets. The excitation-contraction characteristics of these myocytes have not been investigated. Cardiospecific overexpression of calsequestrin also induces hypertrophy.29 156 Myocytes from these mice show reduced CICR from the SR, increased storage of Ca2+ in the SR, and depressed contractility. In contrast, the phospholamban knockout mouse shows positive inotropism and lusitropism (presumably because of release of the phospholamban-mediated inhibition of SERCA2 activity), but hearts are not hypertrophied.157 Equally, overexpression of SERCA2 increases cardiac contraction and relaxation but does not cause hypertrophy.28 However, reductions in cytoplasmic Ca2+ concentrations do not appear to account for the hypertrophy and hyperplasia seen in the CaM transgenic mice, since overexpression of a mutant CaM that binds Ca2+ normally but is defective in signaling to CaM targets/effectors does not induce growth responses.126 Obviously, it would be desirable to characterize Ca2+ handling in all of these transgenic models under uniform conditions, but it is unlikely that this will ever be achieved.
Is there any evidence of interaction between calcineurin, the immunophilins, and proteins involved in excitation-contraction coupling? It is believed that FKBP12 interacts with RyR1, whereas the closely related protein FKBP12.6 interacts with cardiac RyR2 (reviewed in Reference 158158 ). FKBPs may mediate anchoring of calcineurin to the RyR, and this interaction may be involved in the regulation of channel activity through phosphorylation/dephosphorylation (reviewed in Reference 9494 ). FK-506 disrupts this interaction. Interestingly, single RyR2 opening probability was increased in transgenic FKBP12 knockout mice,159 although it is not clear whether increased RyR2 opening results from the absence of FKBP12 alone or the (presumed) absence of interaction of RyR2 with calcineurin. Although the FKBP12 knockout was frequently embryonically lethal, with mice displaying severe cardiac defects, 1 mouse that survived to adulthood showed signs of heart failure and cardiomyopathy.
In addition to release through the RyR, release of [Ca2+]i from stores in the endoplasmic reticulum is regulated by the InsP3 receptor (InsP3R), which is closely related to the RyR. Although the InsP3R was initially thought to be of little importance in the cardiac myocyte (and this is probably true for regulation of contraction), more detailed studies have established the presence of InsP3R species in cardiac myocytes, and at least in the ferret myocyte, the type 2 InsP3R predominates.160 FKBP12 interacts with the InsP3R1 subtype, but it is not known whether InsP3R2 (or the InsP3R3) interacts with FKBP. However, the amino acid residues thought to be most important in binding of FKBP to InsP3R are conserved.161 These results suggest that chronic activation of calcineurin could perturb Ca2+ handling, and, if these perturbations led to defects in contraction, hypertrophy might indirectly ensue.
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Mitochondrial Permeability Transition (MPT) Pore, Calcineurin, and Cell Necrosis/Apoptosis |
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Massive influx of [Ca2+]o has long been known to be associated with death of the cardiac myocyte, a highly aerobic cell containing abundant mitochondria. The MPT is a phenomenon that leads to increased mitochondrial membrane permeability and collapse of the inner mitochondrial membrane electrochemical gradient (vital for regeneration of ATP from ADP and Pi). It can be attributed to the opening of nonspecific high-conductance pores (MPT pores) in the inner mitochondrial membrane that allow passage of small molecules of
1.5 kDa (reviewed in References 162
through 164162 163 164 ). The structure of the MPT pore is not understood, but it
is associated with inner-membrane adenine nucleotide
translocase,165 which exchanges intramitochondrial ATP for
cytoplasmic ADP. In the heart, opening of the MPT pore is increased by
oxidative stress and adenine nucleotide depletion,
conditions that are encountered during ischemia/reperfusion.
These situations enhance the sensitivity of the pore to
Ca2+ and increase binding of mitochondrial matrix
cyclophilin to the mitochondrial membrane in a CsA-sensitive manner.
Indeed, administration of CsA preserves mitochondrial integrity and
cardiac function under adverse conditions (see, eg, References 166
through 168166 167 168 ; reviewed in Reference 162162 ), possibly through inhibition of
MPT pore opening. Mitochondrial permeability is intimately related to necrosis and to the mitochondrial pathway of apoptosis (reviewed in References 162 through 164162 163 164 ). Apoptosis of the cardiac myocyte (reviewed in References 169 and 170169 170 ) is an area arousing a great deal of interest currently because of the putative role of the process in the development of heart failure. In the mitochondrial pathway of apoptosis, release of cytochrome c from mitochondria allows formation of a complex between cytochrome c, apoptotic protease activating factor 1, ATP/dATP, and procaspase 9. This causes aggregation of procaspase 9 and its activation through cleavage to produce active caspase 9, which in turn leads to activation of caspases 3, 6, and 7. These caspases then cleave key substrates, leading to apoptosis. The mitochondrial pathway of apoptosis is inhibited by antiapoptotic members of the Bcl-2 family (Bcl-2 itself and Bcl-XL) and is promoted by proapoptotic Bcl-2 family members (Bax and Bad). Although it does not directly allow escape of cytochrome c as such, opening of the MPT pore may be an initiating event in the mitochondrial apoptotic pathway. However, apoptotic situations involving the mitochondrial pathway that do not involve the MPT pore have been identified, and these may be related to the ability of proteins such as Bax to form membrane channels/pores.
Expression of wild-type calcineurin A and B, or of constitutively activated calcineurin A, promotes apoptosis in BHK cells (particularly in association with Ca2+ influx), and this could be opposed by coexpression of Bcl-2,171 with which calcineurin interacts.172 Although calcineurin bound to Bcl-2 retains its phosphatase activity, formation of the complex is thought to localize calcineurin to an intracellular membrane fraction at which it is unable to dephosphorylate NFATs.172 In agreement with the foregoing, CsA and FK-506 inhibit apoptosis in a lymphoma cell line.173 The mechanisms underlying the proapoptotic function of calcineurin have not been elucidated, although it is possible that dephosphorylation of Bad (Bad phosphorylation suppresses its proapoptotic function) could be important (reviewed in Reference 174174 ).
The preceding discussion suggests that some of the findings of
Molkentin et al14 could be related to the ability of
calcineurin and the MPT pore to promote cell death/apoptosis.
Postmortem hearts from calcineurin A(1398) transgenic mice showed
signs of heart failure with dilatation and gross
fibrosis.14 Although the subject of much current debate,
it has been suggested that human heart failure might be associated with
apoptosis of the cardiac myocyte.175 176
Hypothetically, it could be argued that cardiospecific activation of
calcineurin could promote apoptosis of myocytes, and it is this
that leads to heart failure. CsA could prevent calcineurin-dependent
apoptosis and the MPT. Indeed, in endothelial
cells, CsA inhibits apoptosis by preventing mitochondrial
efflux of cytochrome c.177 One major
problem with this hypothesis is that cardiomyocytic expression of
NFAT3317 leads to hypertrophy.14 However,
it is not clear whether the phenotype was as severe as in the
calcineurin A(1398) transgenic animals, and NFATs are known to play
a role in cardiac development.127 More details of the
NFAT3317 model would be of interest.
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General Comments and Future Directions |
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In addition to more detailed work in experimental animals, there is clearly a need to extend the work to humans. This is never easy, as prospective studies are not feasible. By the time samples of hypertrophied myocardium are available, the initiating signals may no longer be detectable. It is not clear whether the calcineurin pathway can stimulate protein synthesis in myocytes, and this should be examined. Several of the studies cited in this review have made use of transgenic mouse models, and some of the problems associated with this approach have been discussed. Inducible transgenic animals may help. In some transgenic mice, cardiac defects and disturbances in excitation-contraction coupling coexist. By interacting with RyR2 or other proteins, calcineurin A(1
398) might
perturb excitation-contraction coupling, indirectly leading to cardiac
hypertrophy and heart failure. The effects of calcineurin
A(1398) on Ca2+ transients and
contractility in transiently transfected myocytes or in
myocytes from the calcineurin A(1398) transgenic animal would be of
interest. Calcineurin and the immunophilins also interact with
signaling systems that have not been discussed here. These include the
transforming growth factor ß receptor (reviewed in Reference 178178 ) and
the protein kinase A anchoring proteins, which are involved in
targeting protein kinase A to specific cellular locations (reviewed in
Reference 179179 ). Given the importance of protein kinase A in regulating
myocardial contractility through modulation of L-type
Ca2+ channel activity (reviewed in Reference 44 ),
this is another potential point of interaction between calcineurin and
Ca2+ handling. It is unlikely that a single signaling pathway is responsible for the overall hypertrophic response. Interaction of the calcineurin pathway with other signaling pathways, such as the MAPK pathways, should be examined. Intuitively, in the absence of more complex regulation, these 2 pathways would appear to be in opposition, 1 (MAPKs) promoting phosphorylation and the other (calcineurin) promoting dephosphorylation. Examination of MAPK involvement in the regulation of the transactivating activity of transcription factors that associate with NFAT would be worthwhile, and in this regard, GATA4 contains 1 strong MAPK recognition sequence at residues 102 to 105 (Pro-Val-Ser-Pro), as well as several (Ser/Thr)–Pro sequences.106 With these thoughts in mind, I suggest that the involvement of calcineurin in myocardial hypertrophy has not been unambiguously established and that there is life after calcineurin for those of us working in this area.
Note Added in Proof
Transgenic mice cardiospecifically expressing constitutively
activated Gq develop cardiac hypertrophy, then dilated
cardiomyopathy and heart failure.202 The effects of CsA in
this model were equivocal. It did not diminish hypertrophy when heart
weight was expressed relative to body weight, but did so when heart
weight was expressed relative to tibial length. There was also an
indication that CsA prevented some (but not all) of the changes in gene
expression associated with hypertrophy in this model.
Received October 28, 1998; accepted January 25, 1999.
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