| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2001;104:9.)
© 2001 American Heart Association, Inc.
Editorials |
Calcineurin Inhibition in Hypertrophy
Back From the Dead!
From the Division of Cardiology, University of Cincinnati, Cincinnati, Ohio.
Correspondence to Gerald W. Dorn II, MD, Division of Cardiology, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0542. E-mail dorngw{at}ucmail.uc.edu
Key Words: Editorials • cardiomyopathy • hypertrophy • inhibitors • calcineurin
Reactive cardiac hypertrophy has long been recognized as a compensatory process that enhances ventricular function through normalization of wall stress in the face of increased hemodynamic load. For almost as long, it has also been appreciated that the natural history of a hypertrophied heart faced with unremitting hemodynamic overload is progression from a state of functional compensation to one of functional decompensation, or heart failure.1 Under these circumstances, the heart dilates and fails because hypertrophy fails, and in this manner, nature’s temporary cure for hemodynamic overload becomes part and parcel of the disease.
Because a stimulus for hypertrophy is the initiator event in a reactive compensatory response leading to pathological decompensation, an obvious remedy for pathological myocardial hypertrophy is elimination of the stimulus. This is not always possible, however, owing to insufficient control of blood pressure in hypertensive patients, uncorrected valvular stenoses or insufficiencies, or hypertrophy of viable myocardium in postinfarction patients. Furthermore, genetic causes of hypertrophic cardiomyopathy are not directly addressable with current technologies. Therefore, identification of molecular and biochemical mediators of myocardial hypertrophy has been pursued to delineate hypertrophy signaling events that might be susceptible to targeted inhibition. Various candidate signaling pathways, mostly involving hormone receptors, associated G proteins, or their downstream kinase effectors, have been implicated with the use of in vitro and in vivo systems.2 Recently, protein phosphatases have also received attention in this regard.
The most thoroughly investigated and controversial protein phosphatase proposed to be a hypertrophy signaling factor is calcineurin (CN). CN is a ubiquitous phosphatase best known for its effects on T cell–mediated immunity. In these cells and others, sustained elevations in intracellular calcium activate CN, resulting in dephosphorylation of a class of transcription factors, nuclear factors of activated T cells (NFATs), which then translocate to cell nuclei and regulate expression of specific genes. CN is the target for 2 powerful immunosuppressive agents, cyclosporin A (CsA) and FK506, which have helped to revolutionize cardiac transplantation. Its role as a mediator of myocardial hypertrophy was unsuspected, however, until Molkentin et al3 identified CN in this context by using a yeast 2-hybrid screen for upstream mediators of cardiac GATA4 transcription factor activity. These investigators demonstrated that CN was capable of causing hypertrophy by transgenically expressing a constitutively active CN mutant (caCN) in the mouse heart. Striking cardiac hypertrophy developed by a few weeks of age, with progression to dilated cardiomyopathy and heart failure at 3 months.3 It was suggested that CN activation was not only sufficient to cause hypertrophy but also necessary for hypertrophy development because pharmacological inhibition with CsA and FK506 attenuated this response in angiotensin II– or phenylephrine-treated cultured cardiomyocytes and caCN mice.3 In a follow-up study, this same group of investigators showed that CsA and FK506 attenuated hypertrophy development in several (but not all) transgenic models of cardiac hypertrophy, as well as in aorta-banded rats.4
The above findings set off a flurry of studies in which CsA, FK506, or both were used to determine the role of CN signaling in various cardiac hypertrophy models. This level of investigative activity was likely prompted not only by the dramatic results reported by the Molkentin and Olson laboratories3 4 (and the obvious importance and potential clinical relevance of those findings, if true) but also by the relative ease with which such pharmacological inhibition experiments could be performed. The results of these studies have been reviewed in detail5 but can be summarized as being conflicting and, at times, contradictory. The confusion created by pharmacological CN inhibition studies has been attributed to technical variability, but it cannot be overlooked that the agents used to "specifically target" CN proved to be toxic at the levels used and that this toxicity must have confounded interpretation of the data. Thus, it has remained unclear whether CN is a key signaling intermediary in reactive myocardial hypertrophy or whether it is simply one of a growing number of signaling factors that are capable of causing hypertrophy when their activity in the myocardium is unrestrained.
Clarity on this issue required a new approach to CN
inhibition, one that achieved specific functional inhibition without
causing collateral systemic pathology. Recently published in vitro
studies with the noncompetitive CN inhibitory peptides
Cain and AKAP79 demonstrated that CN inhibition by expressed
proteins could prevent agonist-stimulated hypertrophy of
cultured
cardiomyocytes,6
suggesting the potential of a genetic approach. Now, in the span of a
few months, 3 independent studies have been published that used the
cleaner experimental design of transgenically expressing
CN-inhibitory proteins in the mouse
heart.7 8 9
Revisiting the role of CN in in vivo myocardial hypertrophy
in this manner has substantially demystified its effects on myocardial
growth.
In the current issue of
Circulation, Zou and
coworkers7 describe the
effects of cardiac-specific transgenic expression of a dominant
inhibitory CN mutant (dnCN) on pressure-overload
hypertrophy in abdominal aorta–banded mice. In striking
contrast to in vivo inhibition of CN with CsA, the dnCN transgene had
no detrimental effects on unoperated mice. After aortic banding,
nontransgenic mice exhibited increased cardiac CN activity (
2-fold
over baseline), confirming the association between
hemodynamic stress, development of
hypertrophy, and CN activation. This increase in CN
activity was reduced by approximately two thirds in the aorta-banded
dnCN mice, demonstrating in vivo inhibition of myocardial CN activity
by the transgene. In a highly symmetrical experimental result, 3 weeks
after banding there were also 60% to 70% decreases in several
measures of cardiac hypertrophy,
echocardiographic septal and posterior wall
thicknesses, heart weight indexed to body weight,
cardiomyocyte diameter, and the extent of left
ventricular fibrosis. Interestingly, the characteristic
elevations in atrial natriuretic peptide, brain
natriuretic peptide, and
c-fos gene expression seen with
pressure overload were attenuated in the dnCN mice, but no increases in
-skeletal actin and c-jun
mRNA were noted. Myocardial expression of dnCN was thus effective in
inhibiting cardiac hypertrophy at the whole-organ,
cellular, and molecular levels 3 weeks after short-term imposition of a
pressure-overload stimulus.
A similar transgenic approach, with different
CN-inhibitory peptides, was recently reported by De Windt
et al8 in the
Proceedings of the National Academy of
Sciences USA. Two
transgenic mouse strains were created, which expressed the Cain and
AKAP79 peptides previously shown to inhibit
angiotensin- and phenylephrine-stimulated
hypertrophy of cultured neonatal rat
cardiomyocytes.6
Only mice with very low transgene copy numbers were viable, possibly
due to inhibition of normal early postnatal developmental myocardial
growth in mice with greater expression of the CN inhibitor.
However, single- and double-transgene-copy Cain- and
AKAP79-transgenic mice were apparently normal at baseline.
Hypertrophy was induced by long-term infusion of the
ß-adrenergic agonist isoproterenol or by abdominal aorta banding. In
nontransgenic mice challenged in this manner, CN activity was increased
and myocardial hypertrophy developed, with an 25%
increase in heart weight indexed to body weight. In Cain mice, the
normal increase in CN activity after isoproterenol infusion was
virtually abolished; in AKAP79 mice, CN activity was halved after
pressure overloading. Thus, these 2 CN-inhibitory peptides
demonstrated the anticipated biochemical activities when expressed in
myocardium. Transgenic cardiac expression of both CN
inhibitors blunted the isoproterenol-mediated cardiac
hypertrophy by 50% in both mouse strains. Differences
in the extent of hypertrophy inhibition by the 2 peptides
were observed 14 days after pressure overloading; however, whereas
Cain diminished hypertrophy by 70%, AKAP79
reduced it by only 25% to 38%. Thus, transgenic expression of peptide
inhibitors of CN was highly effective in preventing CN
activation after hormonal or mechanical stress and was partially
effective in preventing the early hypertrophic response.
An especially strong feature of the report by De Windt et
al8 is the use of in vivo
adenoviral infection of rat myocardium to assess the
effects of Cain, independent of developmental perturbations that are
inevitable with transgenic expression that uses the -myosin
heavy-chain promoter. In effect, rats were treated with adenoviral
Cain "gene therapy" that targeted CN and then underwent aortic
banding. Seven days after pressure-overload modeling, the normal
increase in CN activity was abolished by adenoviral Cain, and
hypertrophy was diminished by 40%. These results show the
potential for short-term, selective inhibition of CN to modify reactive
myocardial hypertrophy.
In the same issue of the
Proceedings of the National Academy of
Sciences USA, Rothermel et
al9 describe the effects of
transgenic expression of a truncated form of the endogenous
CN-inhibitory protein MCIP1 (myocyte-enriched
CN-inhibitory protein 1). This natural
inhibitor of CN is highly expressed in striated muscle and
is transcriptionally upregulated as a consequence of CN activation.
Thus, MCIP1 represents an endogenous negative
regulatory mechanism for myocyte CN activity. When full-length MCIP1
cDNA was transgenically expressed in myocardium by using
the -myosin heavy-chain promoter, an unexpected RNA splicing event
resulted in expression of a protein missing the amino terminal 80 amino
acids but retaining full CN-inhibitory activity. Mice
expressing lower levels of this protein exhibited a slight decrease in
cardiac mass but were otherwise normal. When crossed with the caCN
mice, the resulting compound-transgenic mice (expressing both
activated CN and the CN inhibitor) were largely
"rescued"; ie, hypertrophy was diminished by
approximately three fourths compared with caCN littermates, and the
characteristic early progression to dilated
cardiomyopathy did not occur. Likewise, premature
mortality in caCN mice was prevented by coexpression of MCIP1. Finally,
MCIP1-transgenic mice had attenuated hypertrophic responses to
long-term isoproterenol infusion and the
physiological hypertrophy stimulus of
unrestrained running. These studies demonstrate that an
endogenous, naturally regulated inhibitor of
myocardial CN signaling is effective in modulating cardiac
hypertrophies resulting from unrestrained CN activity,
catecholamine excess, and exercise.
These 3 nearly simultaneous reports of the
effects of in vivo CN inhibition on cardiac hypertrophy
indicate that CN signaling is necessary for myocardial growth in a
variety of situations, both pathological and
physiological. CN activity was inhibited by
transgenic expression of 4 different proteins/peptides, with remarkably
similar results. At levels of expression that resulted in viable mouse
lines, there was little or no measurable effect of CN inhibition on
baseline cardiac structure or function. Yet an important role for CN in
normal postnatal cardiac developmental growth is strongly suggested by
the dilated cardiomyopathy that occurred in the
higher-expressing Cain
mice8 and by the small
decrease in cardiac mass and inferential evidence of some embryonic
lethality at higher expression levels in MCIP1
mice.9 Furthermore,
inhibition of exercise-induced hypertrophy by MCIP1
supports a role for CN in a physiological adaptive
response of fully developed
hearts.9
On the basis of these in vivo studies, it is difficult to dispute that CN has a critical role in the pathological hypertrophy response to catecholamine excess or pressure overload. Future investigations will determine whether there is a similarly important role in other forms of hypertrophy, particularly hypertrophic cardiomyopathy, wherein the hypertrophy stimulus is an intrinsic genetic defect that may or may not perturb intracellular calcium concentrations.10 It will also be necessary to perform experiments to determine whether hypertrophy signaling through other pathways may ultimately overwhelm inhibition of CN and result in a quantitatively normal but delayed hypertrophic response. Certainly, virtually complete CN inhibition incompletely prevented hypertrophy in one of the studies,8 indicating that CN-independent hypertrophy signaling pathways exist.
If one accepts a central role for CN activation in many forms of cardiac hypertrophy, what then are the therapeutic implications of these studies? It is obvious that CN inhibition can be achieved without the inescapable toxicity afforded by CsA and FK506. As experimental tools these agents were problematic, and as antihypertrophic agents it is not clear that a therapeutic window actually exists. These studies demonstrate that CN can be inhibited by large or small peptides, administered over either the short or long term. However, caution is warranted by the apparent inhibition of normal developmental and physiological hypertrophic responses with "superinhibition" of CN. An ideal "magic bullet" for hypertrophy should eliminate the pathology without ablating the beneficial aspects. After all, hypertrophy is an extremely effective means to diminish chamber wall stress and compensate for diminished intrinsic contractility or increased hemodynamic load; its adverse consequences primarily result from failure of the compensatory mechanism. Future studies will need to address the relative benefits of attenuating or inhibiting hypertrophy versus more subtly altering its fundamental characteristics, perhaps by modulation of signaling events that are not as central to myocardial growth as CN.
Acknowledgments
This study was supported in part by grants HL52318, HL58010, and HL/HD59888 from the National Institutes of Health, Bethesda, Md.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
1.
Meerson
FZ, Pshennikova MG. The mechanism of hypertrophy and wear
of the myocardium. Acta
Cardiol. 1965;20:381–391.
2. Dorn GW II, Brown JH. Gq signaling in cardiac adaptation and maladaptation. Trends Cardiovasc Med. 1999;9:26–34.[Medline] [Order article via Infotrieve]
3. Molkentin JD, Lu JR, Antos CL, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998;93:215–228.[Medline] [Order article via Infotrieve]
4.
Sussman MA, Lim HW,
Gude N, et al. Prevention of cardiac hypertrophy in mice by
calcineurin inhibition.
Science. 1998;281:1690–1693.
5.
Molkentin JD.
Calcineurin and beyond: cardiac hypertrophic signaling.
Circ Res. 2000;87:731–738.
6.
Taigen T, De Windt
LJ, Lim HW, et al. Targeted inhibition of calcineurin prevents
agonist-induced cardiomyocyte hypertrophy.
Proc Natl Acad Sci
U S A. 2000;97:1196–1201.
7.
Zou Y, Hiroi Y,
Uozumi H, et al. Calcineurin plays a critical role in the development
of pressure overload–induced cardiac hypertrophy.
Circulation. 2001;104:97–101.
8.
De Windt LJ,
Lim HW, Bueno OF, et al. Targeted inhibition of calcineurin attenuates
cardiac hypertrophy in
vivo. Proc Natl Acad Sci
U S A. 2001;98:3322–3327.
9.
Rothermel BA,
McKinsey TA, Vega RB, et al. Myocyte-enriched calcineurin-interacting
protein, MCIP1, inhibits cardiac hypertrophy
in vivo.
Proc Natl Acad Sci
U S A. 2001;98:3328–3333.
10. Fatkin D, McConnell BK, Mudd JO, et al. An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy. J Clin Invest. 2000;106:1351–1359.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  J. R. Keys, E. A. Greene, C. J. Cooper, S. V. Naga Prasad, H. A. Rockman, and W. J. Koch Cardiac hypertrophy and altered {beta}-adrenergic signaling in transgenic mice that express the amino terminus of {beta}-ARK1 Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H2201 - H2211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Williams Calcineurin Signaling in Human Cardiac Hypertrophy Circulation, May 14, 2002; 105(19): 2242 - 2243. [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Hill, B. Rothermel, K.-D. Yoo, B. Cabuay, E. Demetroulis, R. M. Weiss, W. Kutschke, R. Bassel-Duby, and R. S. Williams Targeted Inhibition of Calcineurin in Pressure-overload Cardiac Hypertrophy. PRESERVATION OF SYSTOLIC FUNCTION J. Biol. Chem., March 15, 2002; 277(12): 10251 - 10255. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||