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doi:10.1016/j.hfc.2005.03.005
heartfailure.theclinics.com
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Calsequestrin overexpression
CSQ is a high-capacity Ca2+-binding protein in
the junctional SR that forms a quaternary complex
with junctin, triadin, and RyRs. Overexpression of
CSQ in the heart is associated with cardiac hypertrophy [33 36]. Cho and colleagues [33] observed
decreased b-adrenergic receptor (b-AR) responsiveness and density, decreased adenylyl cyclase activity, and increased b-AR kinase 1 (bARK1) levels.
There is less Ca2+-channel gated SR Ca2+ release
and the Ca2+-uptake proteins (Ca2+-ATPase and
PLB) are elevated slightly [34,35]. Proteins involved
in the Ca2+-release cascade (RyRs, junctin, and
triadin) either are downregulated [34] or unchanged
[35]. These findings suggest that CSQ overexpression is associated with increases in SR Ca2+ capacity, but decreases in Ca2+-induced SR Ca2+ release,
leading to depressed contractility in the mammalian heart [35,36] (see rescue of calsequestrinoverexpressing mice with phospholamban knockout
later in this article).
Junctin overexpression
Junctin is a 26-kDa integral membrane protein,
colocalized with RyR, triadin, and CSQ at the
junctional SR membrane in cardiac and skeletal
muscles. The sequence of junctin includes a short
N-terminal cytoplasmic domain, one transmembrane
domain, and a highly charged C-terminal domain
located in the SR lumen. To elucidate the functional
role of junctin in heart, TG mice overexpressing
canine junctin (24- to 29-fold) under the control of
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Triadin 1 overexpression
Triadin 1 is a major transmembrane protein in
cardiac junctional SR that forms a quaternary complex with the RyRs, junctin, and CSQ. TG mice with
targeted overexpression of triadin 1 to mouse atrium
and ventricle have been studied [39]. Triadin 1 overexpression is accompanied by cardiac hypertrophy.
The levels of two other junctional SR proteins, the
RyRs and junctin, are reduced, whereas the levels of
CSQ, PLB, and SERCA2a are unchanged. Cardiac
myocytes from TG mice exhibited depressed contractility, Ca2+ transients decayed at a slower rate, and
cell shortening and relengthening are impaired. The
extent of depression of cell shortening of triadin 1
overexpressing cardiomyocytes is rate-dependent,
being more depressed under low stimulation frequencies, but reaching comparable levels at higher
frequencies of stimulation. Spontaneously beating,
isolated work-performing heart preparations overexpressing triadin 1 also relax at a slower rate than
control hearts, and fail to adapt to increased afterload.
The fast time-inactivation constant of the L-type
Ca2+ channel is prolonged in TG cardiomyocytes.
These findings provide evidence for the coordinated
regulation of junctional SR protein expression in
heart independent of free SR protein expression, and
suggest an important role for triadin 1 in regulating the contractile properties of the heart during
excitation-contraction coupling.
transgenic models of hf
225
Transcription factors
cAMP response element binding protein
overexpression
TG mice overexpressing a dominant-negative
form of the cAMP response element binding protein
(CREB) transcription factor (CREBA133) under the
control of a-MHC promoter develop dilated cardiomyopathy that closely resembles many of the anatomic, physiologic, and clinical features of human
idiopathic dilated cardiomyopathy [49]. These mice
develop four-chamber cardiac dilatation, decreased
systolic and diastolic LV function, and attenuated
contractile responses to the b-adrenergic agonist
isoproterenol. CREBA133 hearts demonstrate atrophic and hypertrophied fibers as well as significant
interstitial fibrosis. Taken together, these results
implicate CREB as an important regulator of cardiac
myocyte function and provide a genetic model of
dilated cardiomyopathy that should facilitate studies
of the pathogenesis and therapy of this clinically
important disorder.
Serum response factor overexpression
Serum response factor (SRF), a member of the
MCM1 (SPELL) family of transcriptional activators,
has been implicated in the transcriptional control of
several cardiac muscle genes, including cardiac
skeletal a-actin and a- and b-MHC. TG mice with
cardiac-specific overexpression of the human SRF
gene [50] develop cardiomyopathy and exhibit
increased heart weight and four-chamber dilation.
Histologic examination reveals cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis.
SRF overexpression alters the expression of SRFregulated genes and results in cardiac muscle
dysfunction. These findings show that sustained overexpression of SRF, in the absence of other stimuli, is
sufficient to induce cardiac abnormalities. SRF is
likely to be a downstream effector of the signaling
pathways involved in mediating cardiac hypertrophy
and failure.
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lar effect in a rat model of pressure-overload hypertrophy [55]. These results suggest that calcineurin
inhibitors merit investigation as potential therapeutics
for certain forms of human heart disease.
TG mice have been developed that express specifically in the heart the calcineurin inhibitory domains
of Cain/Cabin-1 and A-kinase anchoring protein 79
[56]. DCain and DA-kinase-anchoring protein TG
mice demonstrate reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second
approach, adenoviral-mediated gene transfer of
DCain is performed in the adult rat myocardium to
evaluate the effectiveness of an acute intervention and
any potential species dependency. DCain adenoviral
gene transfer inhibits cardiac calcineurin activity and
reduces hypertrophy in response to pressure overload
without reducing aortic pressure. These results
provide genetic evidence implicating calcineurin as
an important mediator of the cardiac hypertrophic
response in vivo.
b-Adrenergic receptor kinase
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knockout mice is increased, although regional distribution of apoptotic cells is normal. Phenotypes of
targeted mutations of many molecules within the
TGF-b signaling pathway (TGF-b1, -b2, -b3, TGF-b
receptor [TGF-bR-II]) and the signaling molecules
SMAD2, SMAD3, and SMAD4 are discussed in a
review [67], although TGF-b2 is the only one with
phenotypes relevant to HF.
Nerve growth factor overexpression
Maintenance of cardiac performance is controlled
tightly by the autonomic nervous system. In congestive heart failure (CHF), although the adverse
pathophysiologic effects of cardiac sympathetic overactivity are recognized increasingly, the paradoxical
finding of reduced sympathetic innervation density in
the failing heart remains unexplained. Nerve growth
factor (NGF) supports the survival of developing
sympathetic and a subpopulation of sensory neurons.
In the adult it participates in maintenance of the
neurotransmitter phenotype of responsive neurons.
The amount of NGF synthesized by a given target
tissue determines its final innervation density; those
developing neurons that fail to receive sufficient NGF
undergo apoptosis.
A TG mouse model has been developed in which
NGF is overexpressed in developing and adult cardiac tissue by placing a NGF minigene under the
transcriptional control of the cardiac-specific a-MHC
promoter [68]. TG mice develop cardiac enlargement
and an increase in myocardial mass. Immunohistochemical analyses with the neural marker S-100
reveal staining of a subpopulation of ectopic cells,
suggesting their derivation from the neural crest.
Another subpopulation of ectopic cells within the
heart expresses neuron-specific enolase. Elevated
cardiac tissue catecholamine levels in TG mice reflect
sympathetic hyperinnervation. Analysis of mediastinal sympathetic ganglia reveals increases in the size
and number of neurons. In this model, increased
expression of NGF produces hyperinnervation of the
heart, pathologic cardiac growth, and the recruitment and expansion of an ectopic, neural, crestderived cell type.
Insulin-like growth factor 1 overexpression
Stimulation of the local renin-angiotensin system
and apoptosis characterize the diabetic heart. Insulinlike growth factor 1 (IGF-1) reduces angiotensin II
and apoptosis. Streptozotocin-induced diabetic cardiomyopathy is attenuated in TG mice overexpressing IGF-1 [69]. Compared with TG, WT mice show
transgenic models of hf
229
lar fibrosis and atrial thrombosis. These findings indicate that production of TNFa by cardiac myocytes
is sufficient to cause severe cardiac disease and
support a causal role for this cytokine in the pathogenesis of human cardiac disease.
Gaq and Gsa overexpression
Receptor-mediated Gq signaling promotes hypertrophic growth of cultured neonatal rat cardiac myocytes and is postulated to transduce in vivo cardiac
pressure overload hypertrophy. Although initially
compensatory, hypertrophy can proceed by unknown
mechanisms to cardiac failure. Expression of a
constitutively activated mutant of Gaq or overexpression of Gaq, which increase Gq signaling, produces
initial hypertrophy, which rapidly progresses to
apoptotic cardiomyocyte death [76,77]. The result
of cardiomyocyte apoptosis is a transition from compensated hypertrophy to a rapidly progressive and
lethal cardiomyopathy. Progression from hypertrophy
to apoptosis in vitro and in vivo is coincident with
activation of p38 and Jun kinases. These data suggest
a mechanism in which moderate levels of Gq signaling stimulate cardiac hypertrophy, whereas high-level
Gq activation results in cardiomyocyte apoptosis. The
identification of a single biochemical stimulus regulating cardiomyocyte growth and death suggests a
plausible mechanism for the progression of compensated hypertrophy to decompensated HF.
The overexpression of cardiac stimulatory G protein a subunit (Gsa) in TG mice results in chronic
endogenous sympathetic stimulation that leads to a
clinical picture of cardiomyopathy [78]. The LV
ejection fraction is reduced in TG mice. When ejection fractions are compared at similar heart rates, the
Gsa mice exhibit a greater LV end-diastolic dimension. Baseline heart rates and arrhythmias are observed to be elevated in TG mice compared with
control mice. Thus mice with Gsa overexpression
exhibit many features of dilated cardiomyopathy.
This study supports the concept that chronic sympathetic stimulation over an extended period of time
(eg, over the life of an animal), is deleterious and may
result in cardiomyopathy (see article by Crosses elsewhere in this issue).
b-Adrenergic receptor overexpression
The b-AR signaling system plays a fundamental
role in heart function. Chronic stimulation of cardiac
b1-AR contributes to disease progression and mortality in patients and animal models of HF. TG mice
with cardiac-specific overexpression of b1-AR [79]
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lebeche et al
Crosses
Calsequestrin overexpression hypertrophy is
worsened by Na+-Ca2+ exchanger overexpression
Overexpression of CSQ induces severe cardiac
hypertrophy, whereas overexpression of NCX does
not affect cardiac weight. When both CSQ and NCX
are overexpressed (NCX/CSQ) in mice, however,
severe HF develops [84]. The heart to body weight
ratio is enhanced and the mRNA expression of ANF,
a marker of hypertrophy, is highest in double-TG
mice. In isolated muscle strips, the basal relaxation
time is prolonged in CSQ and NCX/CSQ mice. Furthermore, in the presence of caffeine, force of
contraction is increased only in CSQ and NCX/CSQ.
In some respects, however, overexpression of
NCX alters the CSQ phenotype into the WT phenotype. The expression of SERCA and PLB, proteins
involved in the Ca2+ uptake of the SR, are increased
only in CSQ, indicating a possible influence of
NCX in the regulation of SR-Ca2+ uptake proteins.
The Ca2+ transients and the L-type Ca2+ currents in
the presence of caffeine were large in CSQ, but
smaller increases are noted in double-TG mice.
Therefore, the successful co-overexpression of CSQ
and NCX in these mice provides a novel model in
which to investigate the interaction of proteins tightly
linked to maintain Ca2+ homeostasis.
Rescue of calsequestrin-overexpressing mice with
phospholamban knockout
CSQ is the Ca2+-binding protein of junctional SR.
Cardiac-specific overexpression of murine cardiac
CSQ results in depressed cardiac contractile parameters, low Ca2+-induced Ca2+ release from SR, and
cardiac hypertrophy in TG mice. CSQ-overexpressing mice have been crossbred with PLBKO mice
[85]. PLB ablation in CSQ-overexpressing mice leads
to reversal of the depressed cardiac contractile parameters, which is associated with increases of SR
Ca2+ storage. Prolonged action potentials, ventricular
myocyte size, and expression levels of ANF and
a-skeletal actin mRNA in CSQ-overexpressing cardiomyocytes also reverses to normal upon PLB
ablation. These results indicate that attenuation of
PLB function may prevent or overcome functional
and remodeling defects in hypertrophied hearts.
transgenic models of hf
231
PLB-deficient mice to generate a model with SERCA1a overexpression in the phospholamban null
background (SERCA1+/PLB ) [90]. Quantitative
immunoblotting reveals an increase in total SERCA
level, whereas SERCA2 is decreased compared with
WT. Isolated myocytes indicate increases in the
maximal rates of contraction and maximal rates of
relaxation, whereas the time for 80% decay of the
Ca2+-transient is decreased in SERCA1+/PLB hearts
compared with SERCA1a overexpressors and PLB
knockouts. These mechanical alterations reflect parallel alterations in Vmax and effective concentration50
for Ca2+ of the SR-Ca2+ transport system. Furthermore, the myocyte contractile parameters remain
enhanced up to 12 months of age. These results
indicate that a combination of SERCA1a overexpression and PLB ablation further enhances myocyte
contractility over each individual alteration.
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lebeche et al
vascular resistance
Myocardial function
Disturbed excitation-contraction
coupling
Increased SERCA2a
expression/activity
6 Increased NCX expression
6 Increased PLB
6 Decreased RyR and L-type
Ca2+ channels
6 Altered myofilament
Ca2+ sensitivity
Altered adrenergic receptor signaling
Increase in connective tissue content
(at the expense of myosin)
6
Molecular
Reactivation of embryonic
Summary
Genetic animal models of HF are used widely and
are critically important for exploring and under-
gene program
Increased ANF mRNA
6 Increased B-skeletal actin mRNA
Increased C-MHC
6
transgenic models of hf
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
References
[1] Smith HJ, Nuttall A. Experimental models of heart
failure. Cardiovasc Res 1985;19:181 6.
[2] Gwathmey JK. Experimental cardiomyopathies. In:
Braunwald E, Abelman WH, editors. Atlas of
heart disease: cardiomyopathies, myocarditis, pericardial disease. Philadelphia7 Current Medicine; 1994.
p. 11.1 11.21.
[3] Hongo M, Royke T, Ross J. Animal models of heart
failure: recent developments and perspectives. Trends
Cardiovasc Med 1997;7:161 7.
[4] Hasenfuss G. Animal models of human cardiovascular
disease, heart failure and hypertrophy. Cardiovasc Res
1998;39:60 76.
[5] James J, Hewett T, Robbins J. Cardiac physiology in
transgenic mice. Circ Res 1998;82:407 15.
[6] Sugawara M, Kato K, Komatsu M, et al. A novel de
novo mutation in the desmin gene causes desmin myopathy with toxic aggregates. Neurology 2000;55(7):
986 90.
[7] Wang X, Osinska H, Dorn 2nd GW, et al. Mouse
model of desmin-related cardiomyopathy. Circulation
2001;103(19):2402 7.
[8] Sussman MA, Welch S, Cambon N, et al. Myofibril
degeneration caused by tropomodulin overexpression
[18]
[19]
[20]
[21]
[22]
[23]
[24]
233
234
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
lebeche et al
and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic
stenosis. Cardiovasc Res 2002;53(2):372 81.
Brittsan AG, Carr AN, Schmidt AG, et al. Maximal
inhibition of SERCA2 Ca(2 +) affinity by phospholamban in transgenic hearts overexpressing a nonphosphorylatable form of phospholamban. J Biol
Chem 2000;275(16):12129 35.
Li Y, Kranias EG, Mignery GA, et al. Protein kinase A
phosphorylation of the ryanodine receptor does not
affect calcium sparks in mouse ventricular myocytes.
Circ Res 2002;90(3):309 16.
Kadambi VJ, Ponniah S, Harrer JM, et al. Cardiacspecific overexpression of phospholamban alters
calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice. J Clin Invest 1996;97(2):
533 9.
Loukianov E, Ji Y, Grupp IL, et al. Enhanced myocardial contractility and increased Ca2 + transport
function in transgenic hearts expressing the fast-twitch
skeletal muscle sarcoplasmic reticulum Ca2 + -ATPase.
Circ Res 1998;83(9):889 97.
Ji Y, Loukianov E, Loukianova T, et al. SERCA1a
can functionally substitute for SERCA2a in the heart.
Am J Physiol 1999;276(1 Pt 2):H89 97.
He H, Giordano FJ, Hilal-Dandan R, et al. Overexpression of the rat sarcoplasmic reticulum Ca2 +
ATPase gene in the heart of transgenic mice accelerates
calcium transients and cardiac relaxation. J Clin Invest
1997;100(2):380 9.
East JM. Sarco(endo)plasmic reticulum calcium
pumps: recent advances in our understanding of
structure/function and biology. Mol Membr Biol
2000;17(4):189 200 [review].
Frank KF, Bolck B, Brixius K, et al. Modulation of
SERCA: implications for the failing human heart.
Basic Res Cardiol 2002;97(Suppl 1):I72 8.
Cho MC, Rapacciuolo A, Koch WJ, et al. Defective
b-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice
with calsequestrin overexpression. J Biol Chem 1999;
274:22251 6.
Jones LR, Suzuki YJ, Wang W, et al. Regulation of
Ca2+ signaling in transgenic mouse cardiac myocytes
overexpressing calsequestrin. J Clin Invest 1998;101:
1385 93.
Sato Y, Ferguson DG, Sako H, et al. Cardiac-specific
overexpression of mouse cardiac calsequestrin is
associated with depressed cardiovascular function and
hypertrophy in transgenic mice. J Biol Chem 1998;
273:28470 7.
Schmidt AG, Kadambi VJ, Ball N, et al. Cardiacspecific overexpression of calsequestrin results in left
ventricular hypertrophy, depressed force-frequency
relation and pulsus alternans in vivo. J Mol Cell
Cardiol 2000;32(9):1735 44.
Hong CS, Cho MC, Kwak YG, et al. Cardiac remodeling and atrial fibrillation in transgenic mice overexpressing junctin. FASEB J 2002;16(10):1310 2.
transgenic models of hf
[53] Klee CB, Ren H, Wang X. Regulation of the calmodulin-stimulated protein phosphatase, calcineurin.
J Biol Chem 1998;273(22):13367 70.
[54] Lim HW, De Windt LJ, Mante J, et al. Reversal of
cardiac hypertrophy in transgenic disease models
by calcineurin inhibition. J Mol Cell Cardiol 2000;
32(4):697 709.
[55] Sussman MA, Lim HW, Gude N, et al. Prevention
of cardiac hypertrophy in mice by calcineurin inhibition. Science 1998;281(5383):1690 3.
[56] De Windt LJ, Lim HW, Bueno OF, et al. Targeted
inhibition of calcineurin attenuates cardiac hypertrophy
in vivo. Proc Natl Acad Sci USA 2001;98(6):3322 7.
[57] Koch WJ, Rockman HA, Samama P, et al. Cardiac
function in mice overexpressing the beta-adrenergic
receptor kinase or a beta ARK inhibitor. Science 1995;
268:1350 3.
[58] Koch WJ, Rockman HA. Exploring the role of the
beta-adrenergic receptor kinase in cardiac disease
using gene- targeted mice. Trends Cardiovasc Med
1999;9(3 4):77 81 [review].
[59] Shioi T, Kang PM, Douglas PS, et al. The conserved
phosphoinositide 3-kinase pathway determines heart
size in mice. EMBO J 2000;19(11):2537 48.
[60] Shioi T, McMullen JR, Kang PM, et al. Akt/protein
kinase B promotes organ growth in transgenic mice.
Mol Cell Biol 2002;22(8):2799 809.
[61] Matsui T, Li L, Wu JC, et al. Phenotypic spectrum
caused by transgenic overexpression of activated Akt
in the heart. J Biol Chem 2002;277(25):22896 901.
[62] Naga Prasad SV, Esposito G, Mao L, et al. Gbgdependent phosphoinositide 3-kinase activation in
hearts with in vivo pressure overload hypertrophy.
J Biol Chem 2000;275(7):4693 8.
[63] Wu Y, Temple J, Zhang R, et al. Calmodulin kinase II
and arrhythmias in a mouse model of cardiac hypertrophy. Circulation 2002;106(10):1288 93.
[64] Zeng J, Rudy Y. Early afterdepolarizations in cardiac
myocytes: mechanism and rate dependence. Biophys J
1995;68:949 64.
[65] Alden KJ, Goldspink PH, Ruch SW, et al. Enhancement
of L-type Ca(2+) current from neonatal mouse ventricular myocytes by constitutively active PKC-betaII.
Am J Physiol Cell Physiol 2002;282(4):C768 74.
[66] Bartram U, Molin DG, Wisse LJ, et al. Double-outlet
right ventricle and overriding tricuspid valve reflect
disturbances of looping, myocardialization, endocardial cushion differentiation, and apoptosis in TGFbeta(2)-knockout mice. Circulation 2001;103(22):
2745 52.
[67] Dunker N, Krieglstein K. Targeted mutations of
transforming growth factor-beta genes reveal important
roles in mouse development and adult homeostasis.
Eur J Biochem 2000;267(24):6982 8 [review].
[68] Hassankhani A, Steinhelper ME, Soonpaa MH, et al.
Overexpression of NGF within the heart of transgenic
mice causes hyperinnervation, cardiac enlargement,
and hyperplasia of ectopic cells. Dev Biol 1995;169:
309 21.
235
236
[84]
[85]
[86]
[87]
[88]
lebeche et al
Ca2 + current in transgenic mice with a cardiacdirected expression of the human adenylyl cyclase type
8 (AC8). FASEB J 2002;16(12):1636 8.
Linck B, Boknik P, Huke S, et al. Functional properties
of transgenic mouse hearts overexpressing both calsequestrin and the Na(+)-Ca(2 +) exchanger. J Pharmacol Exp Ther 2000;294(2):648 57.
Sato Y, Kiriazis H, Yatani A, et al. Rescue of
contractile parameters and myocyte hypertrophy in
calsequestrin overexpressing myocardium by phospholamban ablation. J Biol Chem 2001;276(12):9392 9.
Arber S, Hunter JJ, Ross Jr J, et al. MLP-deficient
mice exhibit a disruption of cardiac cytoarchitectural
organization, dilated cardiomyopathy, and heart failure.
Cell 1997;88(3):393 403.
Omens JH, Usyk TP, Li Z, et al. Muscle LIM protein
deficiency leads to alterations in passive ventricular
mechanics. Am J Physiol Heart Circ Physiol 2002;
282(2):H680 7.
Minamisawa S, Hoshijima M, Chu G, et al. Chronic
[89]
[90]
[91]
[92]