Heart Failure Clin 1 (2005) 219 236

Transgenic Models of Heart Failure: Elucidation of the

Molecular Mechanisms of Heart Disease
Djamel Lebeche, PhD*, Rishikesh Dalal, Monica Jang,
Federica del Monte, MD, PhD, Roger J. Hajjar, MD
Massachusetts General Hospital, Charlestown, MA, USA

In recent years, several animal models of heart

failure (HF) have been generated [1 4]. There are
two main types of animal models: naturally occurring models and experimentally induced models
(eg, chronic cardiac pacing, myocardial ischemia,
pressure and volume overload, or genetically altered). This article focuses on the most potentially
and widely used gene-based murine models of cardiac failure.
Transgenic (TG) technology involves the ability
to alter specifically the expression or activity of
known gene products in intact animals [5]. Such
techniques provide a powerful experimental system
to study pathophysiology. Traditional TG approaches
rely on gain-of-function or loss-of-function alterations. Many murine model systems of human heart
disease have been established recently based on these
attributes. The purpose of this article is to identify the
major functional classes of genes that have been
altered and to outline the results of experiments using
these TG models.
Assessment of the cardiovascular phenotype in
the genetically manipulated mouse helps define the
function of a given candidate gene. The choice of
the candidate gene causing a specific cardiovascular
disease presents a particular challenge because
cardiovascular disease is often the result of the
complex interplay of multiple genetic and environ-

mental factors. For some forms of cardiovascular

disease the relative importance of genetic factors is
defined poorly, but for others, genetic factors are
critical. Although some TG models may not have
direct correspondence to identifiable human conditions, they are nevertheless valuable tools for furthering our understanding of the molecular
mechanisms of HF.

Why animal models?

The progress made in our understanding of the
pathophysiology and treatment of HF would not have
been possible without several animal models of HF.
Animal models can be used to address specific
questions not answered easily in patients who have
HF. They allow the design of clinical trials without
having to impose accepted treatment modalities in
addition to the scientific questions being investigated.
They also may serve as a guide to appropriate
therapeutic strategies for human HF because the
primary goal of animal research is its application to
human physiology and disease. The availability of
human myocardium, through cardiac transplantation
programs, makes direct comparison between animal
and human results readily achievable.

Criteria for a model selection

* Corresponding author. Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical
School, CNY-4 149, 13th Street, Charlestown, MA 02129.
E-mail address: dlebeche@partners.org (D. Lebeche).

Because no single animal model can mimic

successfully the human clinical conditions of HF,
several factors should be considered when selecting a

1551-7136/05/$ see front matter D 2005 Elsevier Inc. All rights reserved.
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lebeche et al

model: it should mimic as closely as possible the

human syndrome of HF despite HF having many
underlying causes; it should be sufficiently economical (to permit experiments in many animals); and it
should be accessible, reproducible, relatively stable
under chronic conditions, and suitable for measurement of cardiac size and function. In addition to ease
of experimental manipulations, the results should
provide transferability and extrapolation to the
clinical setting. For great clinical relevance, studies
should be made in conscious animals with intact reflexes.

Genetic models of heart failure

Several genetic models of HF have been reported.
To better understand the relevance of each model,
this article classifies each model according to the
structural/cellular localization or function of the
altered genes.
Myofilament and cytoskelatal disruptions
Desmin-related myopathies (desminopathies)
Desmin is the main intermediate filament (IF)
protein in skeletal and heart muscle cells. The IFs
surround and interlink myofibrils, and connect the
peripheral myofibrils with the sarcolemma. Desminrelated myopathy (DRM) is characterized by abnormal intrasarcoplasmic accumulation of desmin,
potentially associated with a genetic mutation of the
gene encoding for the protein. Two kinds of deletions
and seven missense mutations in the desmin gene
have been identified [6]. To characterize the genetic
defect, several TG mouse lines that expressed either
murine wild-type (WT) desmin or a 7-amino acid
deletion (R173 E179) desmin (D7-des) mutation
linked to DRM have been made [7]. In contrast to
the mouse overexpressing WT desmin, the D7-des
mouse heart shows aberrant intrasarcoplasmic and
electron-dense granular filamentous aggregates that
are desmin-positive and characteristic of human
DRM. The desmin filament network is disrupted,
and myofibril alignment is compromised visibly. The
ability of the heart to respond to b-agonist stimulation
is blunted significantly. Upregulation of desmin
protein at moderate levels is not detrimental, but the
D7-des mutation is dominant negative, and expression of the mutant protein leads to the appearance of
aggregates that are characteristic of and diagnostic for
human desmin-related cardiomyopathy.
Upregulation of ab-crystallin (Cryab), a small
heat shock protein, is associated with various

diseases, including the desmin-related myopathies.

Cryab, which binds to desmin and cytoplasmic actin,
may participate as a chaperone in IF formation and
maintenance. A mutation in Cryab, R120G, has been
linked to a familial desminopathy. Multiple TG
mouse lines have been developed that overexpress
either murine WT Cryab or the R120G mutation [7].
Overexpression of WT Cryab is benign, with no
increases in mortality and no induction of desminrelated cardiomyopathy. In contrast, lines expressing
the R120G mutation are compromised, with a highexpressing line exhibiting 100% mortality by early
adulthood. Modest expression levels result in a
phenotype that was strikingly similar to that observed
for the desmin-related cardiomyopathies. The desmin
filaments in the cardiomyocytes are affected overtly,
myofibril alignment is impaired significantly, and a
hypertrophic response occurs at the molecular and
cellular levels. The data show that the R120G mutation that causes a desminopathy is dominant negative and results in cardiac hypertrophy.
Tropomodulin overexpression
Loss of myofibril organization is a common
feature of chronic dilated and progressive cardiomyopathy. To study how the heart compensates for
myofibril degeneration, TG mice that undergo progressive loss of myofibrils after birth were created
[8]. Myofibril degeneration was induced by overexpression of tropomodulin, a component of the thin
filament complex that determines and maintains
sarcomeric actin filament length. The tropomodulin
cDNA has been placed under control of the cardiacspecific a-myosin heavy chain (a-MHC) gene
promoter [9] to overexpress tropomodulin specifically in the myocardium. Hearts from these mice
present characteristics consistent with dilated cardiomyopathy and failing hypertrophic response. Histologic analysis shows widespread loss of myofibril
organization. Confocal microscopy of isolated cardiomyocytes reveals intense tropomodulin immunoreactivity in TG mice with abnormal coincidence of
tropomodulin and a-actinin reactivity at Z discs. This
novel experimentally induced cardiomyopathy will
be useful for further understanding the pathogenesis
of dilated cardiomyopathy and the effect of thin
filament based myofibril degeneration on cardiac
structure and function.
Cardiac troponin knockout
Troponin I is a subunit of the thin filament
associated troponin-tropomyosin complex involved
in Ca2+ regulation of skeletal and cardiac muscle
contraction. Familial hypertrophic cardiomyopathy

transgenic models of hf

can be caused by mutations in the genes for b-MHC,

a-tropomyosin, cardiac troponin T, or troponin I. The
cardiac isoform of troponin I has been deleted by
using gene targeting in murine embryonic stem cells
to determine the developmental and physiologic
effects of the absence of this regulatory protein
[10]. Mice with a deletion of cardiac troponin I are
born healthy, with normal heart and body weight,
because a fetal troponin I isoform (identical to slow
skeletal troponin I) compensates for the absence of
cardiac troponin I. Compensation is only temporary;
15 days after birth, slow skeletal troponin I expression begins a steady decline, giving rise to a troponin I deficiency. Mice die of acute HF on day 18,
demonstrating that some form of troponin I is required for normal cardiac function and survival. Ventricular myocytes isolated from these troponin I
depleted hearts display shortened sarcomeres and
elevated resting tension measured under relaxing
conditions and have a reduced myofilament Ca2+
sensitivity under activating conditions. The results
show that developmental downregulation of slow
skeletal troponin I occurs in the absence of cardiac
troponin I, and the resultant troponin I depletion alters
specific mechanical properties of the myocardium,
leading to a lethal form of acute HF (crossbreeding
phospholamban knockout mice with troponin I variants later in this article).
Sarcomere protein gene mutation models of familial
hypertrophic cardiomyopathy
Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy
(FHC), a disease characterized by left-ventricular
(LV) hypertrophy, angina, and dyspnea that can result in sudden death. A mouse model of FHC has
been generated by the introduction of an R403Q
mutation into the a-cardiac MHC gene. Cardiac histopathology and dysfunction in the a-MHC(403/+)
mice resembles human FHC. Cardiac dysfunction
precedes histopathologic changes, myocyte disarray,
hypertrophy, and age-related fibrosis [11].
When a-MHC(403/+) mice are treated with factors that cause hypertrophy (calcineurin inhibitors
or a K+-channel agonist), they develop accentuated
hypertrophy, worsened histopathology, and show increased risk for early death [12]. Despite distinct
pharmacologic targets, each agent augments diastolic
Ca 2+ concentrations in WT cardiac myocytes,
whereas a-MHC(403/+) myocytes consistently failed
to respond. Pretreatment with a Ca2+-channel antagonist abrogate diastolic Ca2+ changes in WT myocytes and prevent the exaggerated hypertrophic
response of treated a-MHC(403/+) mice, suggesting

221

that FHC-causing sarcomere protein gene mutations

cause abnormal Ca2+ responses that initiate hypertrophic response (see overexpression of Gsa causes
synergistic effects on a model of familial hypertrophic cardiomyopathy later in this article).
Myosin-binding protein C mutation models of
familial hypertrophic cardiomyopathy
FHC is an autosomal dominant disease resulting
from inherited sarcomeric dysfunction. In addition
to a- and b-MHC, cardiac troponin T, and a-tropomyosin, myosin-binding protein C (MyBP-C) genes
recently have been implicated in FHC. MyBP-C is a
substantial component of the myofibrils that interacts with several proteins of the thick filament of
the sarcomere. Multiple mutations within the gene
for cardiac MyBP-C, one of three known isoforms,
have been linked to FHC.
Although sarcomere protein gene mutations cause
FHC, individuals carrying a mutant cardiac MyBP-C
gene usually have a better prognosis than individuals with a-cardiac MHC gene mutations [13].
By 30 weeks of age, a-MHC(403/+) mice demonstrate considerably more LV hypertrophy than
MyBP-C(t/+) mice. Consistent with this finding,
hearts from 50-week-old a-MHC(403/+) mice demonstrate increased expression of molecular markers
of cardiac hypertrophy, but MyBP-C(t/+) hearts do
not demonstrate expression of these molecular
markers until the mice are more than 125 weeks of
age. In addition, cardiac function of a-MHC(403/+)
mice is impaired significantly before the development of LV hypertrophy, whereas cardiac function of
MyBP-C(t/+) mice is not impaired even after the
development of cardiac hypertrophy. Because these
murine counterparts mimic the human FHC, similar
murine models may be useful for predicting the
clinical consequences of other FHC-causing mutations. These data suggest that electrophysiologic and
cardiac function studies may enable more definitive
risk stratification in FHC patients.
A knock-in mouse model that carries N-terminal
shortened cardiac MyBP-C has been generated [14].
The mutant protein was designed to have a similar
size as the skeletal MyBP-C isoforms, whereas known
myosin- and titin-binding sites as well as the
phosphorylatable MyBP-C motif were not altered.
Mutant cardiac MyBP-C is incorporated readily into
the sarcomeres of heterozygous and homozygous
animals and can be phosphorylated by cAMPdependent protein kinase (PKA). Although histologic
characterization of WT and mutant hearts reveals
no differences in phenotype, LV fibers from homozygous mutant mice exhibit an increased Ca2+ sen-

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sitivity of force development, particularly at lower

Ca2+ concentrations, whereas maximal active force
levels remain unchanged. The results suggest that
cMyBP-C may affect myosin-head mobility and that
N-terminal mutations of the protein in FHC could
lead to a hypercontractile state.
To elucidate the role of cardiac MyBP-C in
myocardial structure and function, truncated forms
of MyBP-C that lack the cardiac MHC-binding and
titin-binding domain also have been created [15].
Homozygous mice bearing mutated MyBP-C alleles
are viable but exhibit neonatal onset of a progressive
dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray,
fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice shows LV dilation
and reduced contractile function at birth; myocardial
hypertrophy increases as the animals mature. LV
pressure/volume analysis in adult homozygous mutant mice demonstrates depressed systolic contractility with diastolic dysfunction. These data indicate
the importance of this protein for long-term sarcomere function and normal cardiac morphology. Mice
bearing homozygous FHC-causing mutations may
predict the severity of disease that these mutations
will cause in humans.

Cardiac junctional sarcoplasmic reticulum

Phospholamban
Phospholamban knockout
Phospholamban (PLB) is a key regulator of
cardiac contractility and modulates sarcoplasmic reticulum (SR) Ca2+ sequestration by decreasing the apparent Ca2+ affinity of SR-Ca2+-ATPase (SERCA) in
its dephosphorylated state. Upon PKA-mediated phosphorylation, which is mediated through b-adrenergic
stimulation, the inhibitory effect of PLB on SERCA
activity is relieved [16]. Based on kinetic analysis, it
is believed that PLB decreases the energetic efficiency of the SERCA pump [17].
Ablation of PLB is associated with significantly
enhanced Ca2+ affinity of SERCA and hyperdynamic cardiac function [18 21]. Furthermore, the
enhanced contractility of the PLB-deficient mouse
heart persists with aging [22]. Studies using smooth
muscle cells of cerebral arteries of PLB knockout
(PLBKO) mice found that PLB is critical for frequency modulation of Ca2+ sparks and associated
large-conductance Ca2+-activated K+ channel currents by PKA in smooth muscle [23]. In another

study, hyperdynamic PLBKO hearts were able to

compensate against a sustained aortic stenosis similar
to WT hearts [24] (see mLP knockout and rescue by
phospholamban knowout or bARKct expression,
rescue of calsequestrin-overexpressing mice with
phospholamban knockout, crossbreading phospholamban knockout mice with troponin I variants, and
combined phospholamban ablation and SERCA1a
overexpression later in this article.)
Nonphosphorylatable phospholamban
overexpression
Several lines of TG mice have been generated that
express increasing levels of a nonphosphorylatable
form of PLB (S16A,T17A) specifically in the heart
[25,26]. This PLB double mutant was chosen to
prevent phosphorylation as a compensatory mechanism in vivo. There are no changes in the expression
levels of SERCA2, calsequestrin (CSQ), calreticulin,
and ryanodine receptor (RyR) in TG mice. Assessment of SR-Ca2+ uptake in hearts of TG mice
indicates increases in the inhibition of the affinity
of SERCA2 for Ca2+ with increased PLB expression.
Maximal inhibition is obtained at PLB expression
levels of 2.6-fold or higher. TG hearts with functional
saturation in PLB:SERCA2 ratio (> 2.6:1) exhibit
increases in b-MHC expression, associated with
cardiac hypertrophy. These findings demonstrate that
overexpression of a nonphosphorylatable form of
PLB in TG mouse hearts results in saturation of the
functional PLB:SERCA2 ratio at 2.6:1 and that
approximately 40% of the SR-Ca2+ pumps are
regulated functionally by PLB in vivo [25].
Phospholamban overexpression
To determine whether all the SERCA enzymes
are subject to regulation by PLB in vivo, TG mice
overexpressing PLB in the heart have been generated
[27]. Isolated cardiac myocytes from TG mice exhibit
diminished shortening fraction and decreased rates
of shortening and relengthening. The decreases in
contractile parameters reflect decreases in the amplitude of the Ca2+ signal and prolongation in the time
for decay of the Ca2+ signal, which is associated with
a decrease in the apparent affinity of the SERCA for
Ca2+. In vivo analysis of LV systolic function reveals
decreases in fractional shortening and the normalized
mean velocity of circumferential shortening in TG
mice compared with WT mice. The differences in
contractile parameters and Ca2+ kinetics in TG
cardiomyocytes and the depressed LV systolic function in TG mice are abolished upon isoproterenol
stimulation. These findings show that a fraction of the
Ca2+-ATPases in native SR is not under regulation by

transgenic models of hf

PLB. Expression of additional PLB molecules results

in inhibition of SR Ca2+ transport, decreases in
systolic Ca2+ levels and contractile parameters in
ventricular myocytes, and depression of basal LV
systolic function in vivo.
Sarcoplasmic reticulum Ca2+ transport pump 1
and 2 overexpression
Fast-twitch skeletal muscle SR Ca2+ transport
pump (SERCA1a) is the Ca2+-handling protein of
the free SR. TG mice that specifically overexpress
SERCA1a in the heart using the cardiac-specific
a-MHC promoter have been generated [28]. The
levels of CSQ, PLB, actin, and tropomyosin remained
unchanged in TG mice. The steady-state level of
SERCA phosphoenzyme intermediate and the maximal velocity of Ca2+ uptake is increased, demonstrating that the overexpressed protein is functional.
Although the basal cytosolic Ca2+ signal is decreased
in cardiomyocytes from TG animals, the amplitude
of cytosolic calcium signal and rate of calcium
resequestration is increased. Significantly faster rates
of contraction and relaxation in TG hearts also are
observed. This study demonstrates that the SERCA1a
pump can substitute endogenous SERCA2a functionally, and its overexpression enhances Ca2+ transport
and contractile function of the myocardium.
Another study in which SERCA1a is overexpressed in the heart [29] finds that the maximal
velocity of SR Ca2+ transport is increased approximately twofold in TG hearts, whereas the affinity of
the SERCA pump for Ca2+ is not changed. Addition
of PLB antibody in the Ca2+-uptake assays increases
the apparent affinity for Ca2+ to the same extent in
TG and WT hearts, suggesting that PLB regulates the
SERCA1a pump in TG hearts. The SERCA pump
affinity for ATP, Hill coefficient, pH dependence of
Ca2+ uptake and transport, and rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme)
are similar to those of WT hearts. These findings
further demonstrate that SERCA1a can substitute
functionally for SERCA2a, is regulated by endogenous PLB in the heart, and exhibits several enzymatic
properties similar to those of SERCA2a when
expressed in a cardiac setting.
The Ca2+ ATPase of the SR pump (SERCA2)
plays a dominant role in lowering cytoplasmic Ca2+
levels during cardiac relaxation and reduction of its
activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. TG mice
overexpressing a rat SERCA2 transgene have been
produced [30]. Functional analysis of Ca2+ handling
and contractile parameters in isolated TG cardiac

223

myocytes indicate that the intracellular Ca2+ decline

and myocyte shortening and relengthening are accelerated. Furthermore, TG mice demonstrate significantly accelerated contraction and relaxation that is
augmented further by isoproteronol. These observations demonstrate that increased SERCA2 expression
is feasible in vivo and results in enhanced Ca2+ transients, myocardial contractility, and relaxation that
may have further therapeutic implications (see combined phospholamban ablation and SERCA1a overexpression later in this article.)
Further reading about SERCA structure, function,
and modulation is available in a review article [31],
as is an in-depth article about SERCA modulation
and its implication for correcting HF [32].

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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mouse a-MHC promoter have been generated

[37,38]. Overexpression of the junctin in mouse heart
is associated with heart enlargements, paradoxical
septal motion, impaired LV systolic function, bradycardia, atrial fibrillation, and increased fibrosis.
Overexpression of junctin leads to downregulation
of triadin and RyR and upregulation of dihydropyridine receptor. The L-type Ca2+ current density
and action potential durations increase, which may
cause bradycardia in TG heart. Clearly, overexpression of junctin in the heart leads to substantial cardiac
remodeling and atrial fibrillation.
Junctin overexpression also affects junctional SR
architecture [38]. The width of the junctional SR is
narrower and less variable in TG cells and the CSQ
content is more compact. Another change is the extension of zippered junctional SR domains to nonjunctional regions. A third change is an increase in
the extent of association between SR and T tubules.
The increase in junctin expression affects the packing of CSQ in the junctional SR and facilitates the
association of SR and T tubules.

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.

Ion handling and protein transport

K+-channel overexpression
Action potential duration is prolonged in excitable
cells in many forms of heart disease, often as a result
of reductions in Ca2+-independent transient outward
potassium currents (Ito). TG mice have been generated that express a dominant-negative N-terminal
fragment of the Kv4.2 pore-forming K+-channel
subunit under the control of the mouse a-MHC
promoter [40]. Electrophysiologic analysis demonstrates that Ito density is reduced specifically and the
action potential durations are prolonged in a subset of
TG myocytes. In vivo hemodynamic studies reveal
significant elevations in the mean arterial and peak
systolic ventricular pressures, indicative of enhanced
contractility. By 10 to 12 weeks of age, TG mice
developed clinical and hemodynamic evidence of
congestive HF. Failing TG hearts displayed molecular
and cellular remodeling with evidence of hypertrophy, chamber dilatation, and interstitial fibrosis,
and individual myocytes showed sharp reductions
in Ito and IK1 densities, action potential duration
prolongation, and increased cell capacitance. The results show that Kv4.2 subunits contribute to Ito in
the mouse and demonstrate that manipulation of
cardiac excitability may influence secondarily contractile performance.
Na+-Ca2+exchanger overexpression
Myocytes from failing hearts produce slower and
smaller Ca2+ transients associated with reduction
in expression of SERCA and an overexpression of
Na+-Ca2+ exchanger (NCX) [41,42]. Because the
physiologic role of these proteins is competing for
and removing Ca2+ from the cytoplasm, overexpression of NCX may compensate for less-effective SR
Ca2+ uptake. A comparison of protein concentrations
and functions between ventricular myocytes from TG
mice that overexpress NCX shows increased NCX
expression, while the concentrations of other Ca2+
handling proteins such as SERCA, calsequestrin and
phospholamban were not different [43,44].
In cardiac myocytes isolated from TG mice
overexpressing cardiac NCX the time to peak and
relaxation of twitches and rapid-cooling contractures
is shorter than in WT mice. Ca2+ declines faster in TG
myocytes even without Na+ and Ca2+ in the superfusing solution. This suggests that SR Ca2+ uptake is
faster in these myocytes. The amount of Ca2+ stored
in the SR of TG mouse myocytes is greater than in
WT myocytes, and the increased SR Ca2+ content

transgenic models of hf

may be responsible for the faster rate of SR Ca2+

release and uptake in cells from TG mice [43].
The positive inotropic effect of the Na+-channel
agonist BDF 9148 is pronounced more significantly
in myocardium for TG animals, whereas the inotropic
response to isoprenaline is similar to WT. This
indicates that myocardial function is preserved better
in TG mice after infarction, which results from enhanced SR Ca2+ content through overexpression of
NCX [44]. Five weeks after myocardial infarction, cardiac function is better maintained in TG
mouse hearts overexpressing NCX than in WT
mice [45]. The isometric contractile response to
b-adrenergic stimulation and SERCA content is
greater in papillary muscles from TG than WT mice
(see calcequestrin-overexpression results in hypertrophy, which is worsened by Na+-Ca2+ exchanger
overexpression later in this article).
L-type calcium channel overexpression
The L-type voltage-dependent Ca2+ channel
(L-VDCC) regulates Ca2+ influx in cardiac myocytes
and plays a key role in intracellular Ca2+ homeostasis
and excitation-contraction coupling. The b subunit
of L-VDCC modifies the properties of the channel
complex by allosteric modulation of the b1 subunit
function and by chaperoning the translocation of the
b1 subunit to the plasma membrane. The high-affinity
b subunit-binding domain of the a1 subunit has been
overexpressed in a cardiac-specific manner to act as a
b subunit trap [46 48]. This has the functional effect
of changing the in vivo stoichiometry between the
a1 and b subunits by creating a dominant negative
expression system in a TG mouse model. The predominant b isoform (b2) is located in the membranebound fraction of heart protein, whereas the b1 and
b3 isoforms are mostly cytosolic. There is a significant reduction of b2 in the membrane fraction of the
TG animals, resulting in a decrease in contractility of
the heart and a decrease in L-type Ca2+ current density in myocytes.
Activation of the b-AR pathway causes phosphorylation of the L-VDCC that in turn increases Ca2+
influx. Targeted expression of the L-VDCC b1
subunit in TG mouse ventricles results in marked
blunting of the b-AR pathway [48]. Inotropic and
lusitropic responses to isoproterenol and forskolin
in TG hearts are reduced significantly. Likewise,
Na+/Ca2+ current augmentation induced by isoproterenol and forskolin is depressed markedly in TG
cardiomyocytes despite no change in b-AR. There
may be a pathway for regulation of the b-AR by
Ca2+ channels.

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.

Kinases and phosphatases

Overexpression of active calcineurin
Impaired myocyte calcium handling is a common
characteristic of failing hearts and increases in calcineurin activity, a Ca2+-sensitive phosphatase, have
been implicated in HF phenotype. TG mice with

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cardiac-specific expression of an active form of

calcineurin display depressed function, hypertrophy,
and HF [51,52]. Overexpression of a constitutively
active form of activated calcineurin catalytic subunit
results in significant protein increases in SERCA2
and decreases in PLB, as well as enhanced PLB
phosphorylation [51]. These alterations in the SR
Ca2+-transport proteins result in increased maximum
velocity (Vmax) and Ca2+ affinity of SERCA2. In
contrast, the in vivo cardiac function indicates severely depressed contractility in TG hearts. The
apparent disparity in contractile properties between
the cellular and multicellular preparations may be
caused partially by tissue remodeling, including
interstitial fibrosis and a marked reduction, dephosphorylation, and redistribution of the gap junctional
protein connexin-43, which may compromise intercellular communication. Therefore, despite enhanced
SR Ca2+ handling and contractility in myocytes,
pathologic remodeling and defects in intercellular
coupling may underlie contractile dysfunction of the
calcineurin hearts.

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

Calcineurin inhibitors prevent hypertrophic response

In response to numerous pathologic stimuli, the
myocardium undergoes a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes. Cardiac hypertrophy is
induced by the calcium-dependent phosphatase calcineurin, which dephosphorylates the transcription
factor NF-AT3, enabling it to translocate to the nucleus. NF-AT3 interacts with the cardiac zinc finger
transcription factor GATA4, resulting in synergistic
activation of cardiac transcription [53]. TG mice that
express activated forms of calcineurin or NF-AT3 in
the heart develop cardiac hypertrophy and HF that
mimic the human disease [52].
Pharmacologic inhibition of calcineurin activity
blocks hypertrophy in vivo and in vitro. Cyclosporin
A mediated inhibition of the calcium-regulated
phosphatase, calcineurin (PP2B), reverses cardiac
hypertrophy and myopathic dilation in two TG mouse
models of cardiomyopathy (activated calcineurin
expression and tropomodulin overexpression). In
contrast, a third mouse model of hypertrophic cardiomyopathy caused by activated NFAT3 cardiacspecific expression is not affected by cyclosporin A
[54]. Administration of the calcineurin inhibitors
cyclosporin and FK506 prevents disease in mice that
were predisposed genetically to develop hypertrophic cardiomyopathy as a result of aberrant expression of tropomodulin, myosin light chain-2, or fetal
b-tropomyosin in the heart. Cyclosporin has a simi-

The b-AR signaling system plays a fundamental

role in heart function. Signaling through bARs can be
dampened by the actions of the bARK, a kinase
whose expression and activity are elevated in chronic
human HF. Animals overexpressing bARK1 demonstrate attenuation of isoproterenol-stimulated LV
contractility in vivo, dampening of myocardial adenylyl cyclase activity, and reduced functional coupling of b-AR [57]. Conversely, mice expressing the
b-ARK inhibitor display enhanced cardiac contractility in vivo with or without isoproterenol [57]. A
review by the same author [58] highlights studies that
have used genetically engineered mice to understand
how perturbations in myocardial b-AR signaling can
impact the pathogenesis of cardiac disease adversely.
Interrupting this process may provide novel therapeutic strategies in the treatment of human HF
(see muscle lim protein knockout and rescue by phospholamban knockout or bARKct expression later in
this article).
Phosphoinositide 3 kinase signaling
Two lines of TG mice expressing either constitutively active or dominant-negative mutants of phosphoinositide 3 kinase (PI3K) in the heart have been
developed [59]. Cardiac-specific expression of constitutively active PI3K results in mice with larger
hearts, whereas dominant-negative PI3K results in
mice with smaller hearts. The increase or decrease in

transgenic models of hf

heart size is associated with comparable increase or

decrease in myocyte size. Cardiomyopathic changes,
such as myocyte necrosis, apoptosis, interstitial
fibrosis, or contractile dysfunction, are not observed
in either of the TG mice. Thus, the PI3K pathway
is necessary and sufficient to promote organ growth
in mammals.
Several biochemical studies have suggested that
Akt/protein kinase B is one of the important downstream targets of PI3K. TG mice have been generated expressing constitutively active Akt (caAkt) or
kinase-deficient Akt (kdAkt) specifically in the heart
[60,61]. The heart weight of caAkt TG mice is
increased twofold compared with that of non-TG
mice. The increase in heart size is associated with a
comparable increase in myocyte cell size in caAkt
mice. The kdAkt mutant protein attenuates the
constitutively active PI3K-induced overgrowth of
the heart, and the caAkt mutant protein circumvents
cardiac growth retardation induced by a kinasedeficient PI3K mutant protein. These findings indicate that Akt is sufficient to induce a marked increase
in heart size and is likely to be one of the effectors of
the PI3K pathway in mediating heart growth.
Activation of PI3Ks is coupled to phosphotyrosine/growth factor and G protein coupled receptors.
The role of PI3K activation in myocardium has been
studied during in vivo pressure overload hypertrophy
in mice [62]. Cytosolic extracts from WT hypertrophied hearts showed a selective increase in the
PI3K gamma isoform. TG mice with cardiac-specific
overexpression of a Gbg sequestering peptide have
been used to assess the role of G protein coupled
receptor-mediated activation of PI3K [62]. Extracts
from these hypertrophied TG hearts show complete
loss of PI3K activation, indicating a Gbg-dependent
process. TG mice with cardiac-specific overexpression of a Gq-inhibitor peptide then are used to
determine the class of G proteins that contribute
Gbg dimers for in vivo PI3K activation. Pressureoverloaded Gq-inhibitor TG mice show a complete
absence of PI3K activation, whereas pretreatment
with pertussis toxin shows robust PI3K activation.
These results show that activation of the PI3K during in vivo pressure overload hypertrophy is Gbgdependent and the Gbg dimers arise from stimulation
of Gq-coupled receptors.
Constitutively active calmodulin kinase
overexpression
Calcium-calmodulin dependent protein kinase II
(CaMK) has been implicated in Ca2+-dependent
regulation of L-type Ca2+ current (ICa). CaMK II is

227

linked to arrhythmia mechanisms in cellular models

where repolarization is prolonged. TG mice overexpressing a constitutively active form of CaMKIV
have been created [63]. TG mice have significantly
more arrhythmias than WT mice. Arrhythmias are
increased additionally by isoproterenol and significantly suppressed by an inhibitory agent targeting
endogenous CaMKII. TG mice have longer QT intervals and action potential durations than WT mice,
and TG cardiomyocytes also have frequent early
afterdepolarizations (EADs), a hypothesized mechanism for triggering arrhythmias [64]. L-type Ca2+
channels (LTCCs) can activate EADs, and LTCC
opening probability (Po) was significantly higher in
TG than WT cardiomyocytes before and after isoproterenol [64]. A CaMKII inhibitory peptide equalizes TG and WT LTCC Po and eliminates EADs,
whereas a peptide antagonist of NCX current, also
hypothesized to support EADs, is ineffective [63].
These findings support the hypothesis that CaMKII
is a proarrhythmic signaling molecule in cardiac hypertrophy in vivo.
Constitutively active protein kinase-bII
The cardiac ICa can be modified by activation of
protein kinase C (PKC). A TG mouse that expresses
constitutively active PKC-bII shows a twofold
increase in nifedipine-sensitive ICa [65]. A PKC-bII
antagonist returns ICa amplitude to levels found in
non PKC-bII-expressing myocytes. The increase in
ICa is independent of Cav1.2-subunit mRNA levels. Thus these data demonstrate that this isoform of
PKC-b is an important modulator of cardiac L-type
Ca2+ channels.

Growth factors, cytokines, and receptors

Transforming growth factor b2 knockout
Transforming growth factors b (TGF-b) are
multifunctional molecules with profound biologic
effects in many developmental processes, including
regulation of cell proliferation, differentiation, cell
adhesion, skeletal development, haematopoiesis, inflammatory responses, and wound healing. Mice
deficient in the TGF-b2 gene die around birth and
show various defects of different organs, including
the heart [66]. Hearts of TGF-b2 null mouse embryos from 11.5 to 18.5 days of gestation reveal
multiple defects such as malformations of the atrioventricular canal and outflow tract, and anomalies of
the aorta and its branches. Apoptosis in TGF-b2

228

lebeche et al

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

progressively depressed ventricular performance,

twofold higher myocyte apoptosis, greater loss of
ventricular myocytes, and myocyte hypertrophy. WT
mice also show increases in p53, Bax, angiotensinogen, angiotensin type-1 (AT1) receptors, and
angiotensin II. These findings show that IGF-1
interferes with the development of diabetic myopathy
by attenuating p53 function and angiotensin II
production and thus AT1 activation. The latter event
might be responsible for the decrease in oxidative
stress and myocyte death by IGF-1.
IGF-1 opposes the stimulation of myocyte death
in the surviving myocardium after infarction [70,71].
Compared with mice constitutively overexpressing
IGF-1, nontransgenics show greater myocyte apoptosis and necrosis after coronary ligation. Nontransgenics showed larger decreases in wall thickness,
larger increases in chamber diameter, and increased
chamber volume. Constitutive overexpression of
IGF-1 prevents activation of cell death in the viable
myocardium after infarction, limiting ventricular dilation, myocardial loading, and cardiac hypertrophy.
TG mice overexpressing IGF-1B demonstrate
increased heart weight and more myocytes in the
heart starting 75 days after birth [72]. In contrast,
myocyte cell volume is comparable in TG and control
mice at all ages. In conclusion, overexpression of
IGF-1 in myocytes leads to cardiomegaly mediated
by more cells in the heart.
Retinoic acid receptor overexpression
Retinoids play a critical role in cardiac morphogenesis. TG mice that overexpress a constitutively
active retinoic acid receptor (RAR) controlled by either the a- or b-MHC promoter have been generated
[73]. Animals carrying the a-MHC-RAR transgene
express RARs in embryonic atria and in adult atria
and ventricles, but develop no signs of either
malformations or disease. In contrast, b-MHC-RAR
animals, where expression is activated in fetal ventricles, develop a dilated cardiomyopathy that varies
in severity with transgene copy number. Molecular
markers of hypertrophy, a-skeletal actin, atrial natriuretic factor (ANF), and b-MHC are upregulated.
Thus, expression of a constitutively active RAR in
developing atria or in postnatal ventricles is relatively
benign, whereas ventricular expression during gestation can lead to significant cardiac dysfunction.
gp130 Receptor knockout and constitutive gp130
A ventricular-restricted knockout of the gp130
cytokine receptor through Cre-IoxP mediated re-

transgenic models of hf

combination has been created. Hirota and colleagues

[74] show a gp130-dependent myocyte survival
pathway in the transition to HF. Such conditional
mutant mice have normal cardiac structure and
function, but during aortic pressure overload, these
mice display rapid onset of dilated cardiomyopathy
and massive induction of myocyte apoptosis versus
the control mice, which exhibit compensatory hypertrophy. This suggests that cardiac myocyte apoptosis
is a critical point in the transition between compensatory cardiac hypertrophy and HF. gp130-Dependent
cytokines may represent a novel therapeutic strategy
for preventing in vivo HF.
TG mice having continuously activated gp130
have been created by mating mice from interleukin 6
(IL-6) and IL-6 receptor (IL-6R) TG lines [74]. Offspring overexpressing IL-6 and IL-6R show constitutive tyrosine phosphorylation of gp130 and a
downstream signaling molecule, signal transducer,
and activator of transcription 3 (STAT3). The distinguishing feature of such offspring is hypertrophy of
ventricular myocardium and consequent thickened
ventricular walls of the heart where gp130 is expressed in adulthood. TG mice overexpressing IL-6
or IL-6R alone do not show detectable myocardial
abnormalities. Neonatal heart muscle cells from
normal mice, when cultured in vitro, enlarge in response to a combination of IL-6 and a soluble form of
IL-6R. These results, along with those from the
gp130 knockout mouse indicate that activation of the
gp130 signaling pathways leads to cardiac hypertrophy and that these signals might be involved in
physiologic regulation of myocardium.
Tumor necrosis factor a overexpression
Tumor necrosis factor a (TNFa) is a multifunctional cytokine that has been detected in several human cardiac-related conditions, including CHF and
septic cardiomyopathy. In these conditions, the origin
of TNFa secretion is at least partly from cardiac
myocytes. TG mice in which overexpression of
TNFa is driven by the murine a-MHC promoter
have been developed [75]. Compared with non-TG
mice, TG mice show tachypnea, less activity, and
hunched posture. TG mice document a severe impairment of cardiac function, evidenced by biventricular
dilatation and depressed ejection fractions. All TG
mice died prematurely. Pathologic examination of
affected animals reveals a globular dilated heart,
bilateral pleural effusions, myocyte apoptosis, and
transmural myocarditis in the right and left ventricular free walls, septum, and atrial chambers. In all
terminally ill animals, there is significant biventricu-

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

or constitutively active mutant a1 b-AR [80] have

been created to search for the mechanism of adrenergic impairment of cardiac function in vivo. TG
mice with cardiac overexpression of b1-AR show
progressive LV fibrosis starting at 4 months of age
[79]. TG animals demonstrate cardiac-specific
expression of this a1-AR with resultant activation
of phospholipase C, as shown by increased myocardial diacylglycerol content. A phenotype consistent with cardiac hypertrophy develops in adult TG
mice with increased heart to body weight ratios,
myocyte cross-sectional areas, and ventricular ANF
mRNA levels relative to non-TG controls [80].
Isolated cardiomyocytes from these animals display
markedly altered calcium transients with significant
prolongation of the intracellular calcium transient
compared with non-TG littermates. SR proteins
involved in calcium handling (CSQ, triadin, and
PLB) are not altered in quantity, but junctin progressively decreases.
Signaling through b-ARs can be dampened by
the actions of b-ARK, whose expression and activity
are elevated in chronic human HF. Several reviews
have compiled extensive information about TG or
knockout models of b-ARs or bARKs [58,81,82]. An
overview of b-AR gene regulation, TG models of
bAR overexpression, and a discussion of bAR polymorphisms as they relate to HF progression also
are presented.

Adenylyl cyclase overexpression

The b-adrenergic cascade is impaired severely in
HF, in part because of reduced activity of the two
dominant cardiac adenylyl cyclase (AC) isoforms,
AC5 and AC6. Therefore, cardiac-directed AC overexpression is a conceivable therapeutic strategy in
HF. A TG mouse line with a cardiac-directed expression of the human AC8 has been created [83].
Unlike AC5 and AC6, which are inhibited by
intracellular Ca2+, AC8 is stimulated by Ca2+-calmodulin. Langendorff-perfused hearts from TG mice
have a twofold higher LV systolic pressure, a faster
heart rate, faster relaxation, and higher sensitivity to
external Ca2+ than non-TG mice. The speed of cell
shortening and relaxation in isolated ventricular
myocytes as well as Ca2+ transients is greater in TG
mice. Despite the large increase in Ca2+ transients
and contraction, expression of AC8 has no effect
on the whole-cell ICa amplitude. Moreover, ICa is
unchanged when AC8 is activated by raising intracellular Ca2+. Thus, cardiac expression of AC8 leads
to an increase in cAMP that specifically activates

Ca2+ uptake into the SR but not Ca2+ influx at the

sarcolemma, suggesting a compartmentation of the
cAMP signal.

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

Muscle lim protein knockout and rescue by

phospholamban knockout or b adrenergic receptor
kinase c-terminus expression
Accumulating evidence indicates that cytoskeletal
defects may be an important pathway for dilated
cardiomyopathy and eventual HF. Muscle lim protein
(mLP) is a LIM-only protein of terminally differentiated striated muscle cells, where it accumulates at
actin-based structures involved in cytoarchitecture
organization. mLP-knockout mice develop dilated
cardiomyopathy with hypertrophy and HF after birth
[86]. Analysis reveals dramatic disruption of cardiomyocyte cytoarchitecture. Pressure-volume and pressure-strain relations are altered in mLP-knockout
mice, in general suggesting a less compliant tissue
in the dilated hearts [87].
Dilated cardiomyopathy and end-stage HF result
in multiple defects in cardiac excitation-contraction
coupling. Muscle-specific SERCA2a is inhibited
by PLB. Inhibition of PLB-SERCA2a interaction
through in vivo expression of a PLB point mutant
dominantly activates the contractility of ventricular
muscle cells. The ablation of PLB rescues the spectrum of phenotypes that resemble human HF, including dilated cardiomyopathy [88]. Interfering with the
PLB-SERCA interaction may be a promising therapeutic approach for HF, which is the focus of a
review [89].
HF is accompanied by severely impaired b-AR
function, which includes loss of b-AR density and
functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of
b-AR function is agonist-stimulated receptor phosphorylation by bARK1, an enzyme elevated in failing
heart tissue. bARKct is a c-terminus truncated version
peptide inhibitor of bARK1. In contrast to mLPknockout mice, mLP / /bARKct mice have normal
LV chamber size and function. Furthermore, heightened b-AR desensitization in the mLP-knockout mice,
measured in vivo (responsiveness to isoproterenol)
and in vitro (isoproterenol-stimulated membrane
adenylyl cyclase activity), is reversed completely with
overexpression of the bARK1 inhibitor.

Combined phospholamban ablation and

sarcoplasmic reticulum Ca2+ ATPase
overexpression
PLB ablation or ectopic expression of SERCA1a
in the heart results in significant increases in cardiac
contractile parameters. TG mice with cardiac-specific
overexpression of SERCA1a have been mated with

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.

Crossbreeding phospholamban-knockout mice with

troponin I variants
PLB is a Ca2+-handling protein of the free SR.
b-adrenergic stimulation of the heart results in an
enhanced relaxation rate in association with phosphorylation of cardiac troponin I (cTnI) and PLB.
Four new lines of mice were generated by crossbreeding mice that express slow skeletal troponin I
(ssTnI) with PLBKO mice [91]: PLB/cTnI, PLB/
ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. Perfusion with isoproterenol (ISO) significantly increases
the peak amplitude of fura-2 ratio in PLB/cTnI,
PLBKO/cTnI, and PLBKO/ssTnI groups of mice. In
the presence of ISO, however, there are no differences in the peak amplitude of fura-2 ratio among
cells isolated from hearts of PLB/cTnI, PLBKO/cTnI,
and PLBKO/ssTnI mice. In cells from PLB/cTnI
mice, the extent of shortening is increased, and the
time of relaxation is decreased significantly during
b-adrenergic stimulation. In PLBKO/cTnI cells,
stimulation with ISO increases the extent of shortening and does not change the time of relaxation.
Stimulation with ISO in cells isolated from PLBKO/
ssTnI mice, however, not only significantly increases
the extent of cell shortening but also increases the
time of relaxation. Perfusion with ISO increases
the rate of relaxation only in PLB/cTnI, PLB/ssTnI,
and PLBKO/cTnI muscles, but remains unchanged
in PLBKO/ssTnI muscles. These findings demonstrate that phosphorylation of PLB and cTnI contributes to increased rate of relaxation during
b-adrenergic stimulation.

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lebeche et al

Overexpression of Gsa causes synergistic effects on a

model of familial hypertrophic cardiomyopathy
To determine the effect of chronically enhancing
sympathetic drive, a-MHC(403/+) mice have been
mated with mice overexpressing Gsa to produce
Gsax403 mice [92]. Heart rate in 3-month-old mice is
elevated similarly in mice overexpressing Gsa and
Gsax403 compared with WT and 403 mice. LVEF is
enhanced in Gsax403 mice compared with WT, 403,
and Gsa mice. Isolated cardiomyocytes from Gsax403
mice also exhibited higher baseline percent contraction than WT, 403, and Gsa cardiomyocytes. Relaxation of myocytes was impaired in 403 mice
compared with WT, but enhanced in Gsa and
normalized in Gsax403 mice. This also is observed
in vivo. In-vivo isoproterenol increases LVEF to
maximal levels in Gsax403 and Gsax403, whereas in
403, the response is attenuated compared with WT.
At 10 months of age, Gsax403 has significantly
depressed LVEF. Histopathologic examination demonstrates that myocyte hypertrophy and fibrosis are
present in young Gsax403 mice and that old animals
have severe cardiomyopathy. By 15 months of age,
the survival of Gsax403 mice was 0% compared with
100% for WT, 71% for Gsa, and 100% for 403 mice.
These results show that the cardiomyopathy developed by Gsax403 mice is synergistic rather than
additive, likely owing to the elevated baseline function combined with enhanced responsiveness to sympathetic stimulation.

Box 1. Markers of heart failure

The failing myocardium is characterized by many changes in the autonomic
nervous system and the renin-angiotensin-aldosterone system as well as structural and subcellular abnormalities.
Neurohumoral





Downregulation of C-ARs (density)

G-protein/C-AR uncoupling
High levels of catecholamines
Elevated levels of C-ARK-1 (mRNA
and activity) and G-protein coupled
receptor kinase (mRNA)
 Upregulation of atrial natriuretic peptide
 Increased endothilin-1, tumor
necrosis factor, tumor necrosis
factor-receptor, and IL-6
Hemodynamic
 Elevated filling pressures
 Reduced cardiac output
 Increased pulmonary and systemic

vascular resistance
Myocardial function
 Disturbed excitation-contraction

Gaq and Ga11 double knockout

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

The fact that cardiomyocyte-specific TG overexpression of Gaq results in myocardial hypertrophy

demonstrates that chronic activation of the Gq/G11family is sufficient to induce myocardial hypertrophy.
A mouse line lacking Gaq and Ga11 in cardiomyocytes has been created to test whether Gq/G11
mediates the physiologic hypertrophy response to
pressure overload [77]. These mice show no detectable ventricular hypertrophy in response to pressure
overload induced by aortic constriction. The complete lack of a hypertrophic response shows that the
Gq/G11-mediated pathway is critical for cardiac
hypertrophy induced by pressure overload.

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

standing the molecular mechanisms and determinants

of myocardial failure. A few considerations are to be
kept in mind when designing genetically based
studies: (1) different inducers of HF activate different
signaling pathways, (2) each animal model has advantages and limitations, (3) studies of human heart
tissue and the comparison of normal tissue with failing have led to the identification of several markers
of heart failure (Box 1) and several animal models
share many of these markers, and (4) although the
endpoint (ie, HF) is the same, the pathophysiology
differs between the various animal models. The
findings derived from these animal models of HF
should be interpreted carefully, keeping in mind the
complexity of the human disease process. By merely
altering the gene dosages, one significantly perturbs
the overall stoichiometry of protein production with
subsequent dramatic effects on function. It is possible
that the observed phenotypes are brought about by
the imbalance in the stoichiometry of the expressed
proteins rather than by differences in protein function.
Most experiments in these animal models are performed in young adults despite the fact that HF is
usually a disease of older patients. Although aging,
per se, is not the cause of the cardiomyopathy, there
are age-related alterations in gene expression or
activity signaling pathways.

[9]

[10]

[11]

[12]

[13]

[14]

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