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Review
Sarcomeric dysfunction in heart failure
Nazha Hamdani1, Viola Kooij1, Sabine van Dijk1, Daphne Merkus2, Walter J. Paulus1,
Cris dos Remedios3, Dirk J. Duncker2, Ger J.M. Stienen1, and Jolanda van der Velden1*
1
Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, van der Boechorststraat 7,
1081 BT Amsterdam, The Netherlands;
2
Experimental Cardiology, Thoraxcenter, Cardiovascular Research School COEUR,
Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands; and
3
Muscle Research Unit,
Institute for Biomedical Research, The University of Sydney, Sydney, Australia
Received 30 July 2007; revised 9 October 2007; accepted 24 October 2007; online publish-ahead-of-print 30 November 2007
Time for primary review: 27 days
Sarcomeric dysfunction plays a central role in reduced cardiac pump function in heart failure. This
review focuses on the alterations in sarcomeric proteins in diseased myocardium that range from
altered isoform expression to post-translational protein changes such as proteolysis and phosphoryl-
ation. Recent studies in animal models of heart failure and human failing myocardium converge and
indicate that sarcomeric dysfunction, including altered maximum force development, Ca
2þ
sensitivity,
and increased passive stiffness, largely originates from altered protein phosphorylation, caused by
neurohumoral-induced alterations in the kinase
–
phosphatase balance inside the cardiomyocytes.
Novel therapies, which specifically target phosphorylation sites within sarcomeric proteins or the
kinases and phosphatases involved, might improve cardiac function in heart failure.
KEYWORDS
Sarcomere;
Cardiomyocyte;
Contractility;
Heart failure
1. Sarcomeric dysfunction
The failing heart is characterized by reduced contractility
(systolic dysfunction) and/or impaired filling (diastolic dys-
function). A number of factors, including changes in
cardiac structure (dilation and hypertrophy), apoptotic and
necrotic cell death, maladaptive remodelling of the extra-
cellular matrix, abnormal energy metabolism, impaired
calcium handling, and neurohumoral disturbances have
been implicated in the initiation and progression of heart
failure.
1
–
4
Recent studies revealed that alterations in sarco-
meric function play a prominent role in reduced cardiac
pump function.
Sarcomeric function is determined by the expression
levels of multiple isoforms and by post-translational modifi-
cations of sarcomeric proteins. During muscle contraction a
molecular interaction takes place between the thin (actin)
and thick (myosin) filament of the sarcomeres, which is trig-
gered by a rise in the intracellular calcium and is driven by
the energy from ATP hydrolysis.
5
The tropomyosin
–
troponin
complex inhibits the actin
–
myosin interaction at low intra-
cellular free calcium (Figure 1A). This inhibition is released
when intracellular free calcium increases and binds to
troponin C (Figure 1B). Alterations in sarcomeric protein
composition under pathological conditions will influence
contractile performance of the heart. Within the first part
of this review, we discuss the functional role of individual
sarcomeric protein isoforms and of post-translational
protein modifications such as proteolysis and phosphoryl-
ation. In the second part, we highlight the major changes
in sarcomeric function reported in failing myocardium and
discuss the most likely underlying protein modifications.
2. Isoform composition and sarcomeric
dysfunction
2.1 Myosin heavy chains
The thick filament is composed of myosin, which consists of
two myosin heavy chains (MHC), and two pairs of myosin
light chains (MLCs) (Figure 1). One of the major isoform
changes which has been observed in hypertrophied and
failing ventricular myocardium is the shift from the fast
a-MHC to the slow b-MHC.
6
–
8
The magnitude of the MHC
shift largely depends on the amount of endogenous a-MHC
present in ventricular tissue, which is species-dependent,
being largest in small rodents and smallest in human.
6
–
11
Hence, the functional significance of the shift in MHC com-
position in diseased human ventricles is still a matter of
debate.
8,10,12
The MHCs carry the site for ATP hydrolysis and are import-
ant determinants of the rate of energy consumption and the
speed of contraction of the sarcomeres, which are closely
related.
13
In vitro studies have shown that the a-MHC
isoform has a higher ATPase activity
14
and a higher actin fila-
ment sliding velocity compared with the b-MHC isoform.
15
*Corresponding author. Tel: þ31 20 4448123; fax: þ31 20 4448255.
E-mail address: j.vandervelden@vumc.nl
Published on behalf of the European Society of Cardiology. All rights reserved. &The Author 2007.
For permissions please email: journals.permissions@oxfordjournals.org.
Cardiovascular Research (2008) 77, 649
–
658
doi:10.1093/cvr/cvm079
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From experiments in permeabilized cardiac preparations
from small mammals and humans it followed that the
b-MHC is 3
–
5 times more economical,
11,16,17
but is associ-
ated with reduced power output
18
and shortening vel-
ocity
17,18
compared with the a-MHC isoform. Consequently,
in rats a shift from a-tob-MHC coincided with significant
reductions in ATPase activity,
7,19
tension cost,
19
and power
output.
18
Evidence that a shift in MHC may be of pathophy-
siological relevance for human sarcomeric dysfunction was
already provided in 1962 by Alpert and Gordon
20
who
reported reduced myofibrillar ATPase activity in human con-
gestive heart failure. However, subsequent human studies
failed to unequivocally link this observation with an MHC
isoform shift in failing ventricular myocardium. First, no
differences were found in functional properties of myosin
isolated from non-failing and failing hearts,
10,21,22
indicating
that protein alterations other than a change in myosin com-
position are responsible for the reduction in myofilament
ATPase activity.
20,23
Moreover, various expression levels of
a-MHC (ranging from 0 to 30%) have been reported in the
ventricles of different individuals.
8,10,12,24
This may be due
to age-dependent changes in MHC composition and hetero-
geneous expression of MHC isoforms within the ventricular
wall.
25,26
Both in human
25
and rat
26
ventricular tissue, a
regional difference in MHC expression has been observed,
characterized by a higher expression of the fast a-MHC in
the subepicardial than in the subendocardial layer,
consistent with the shorter action potential and contraction
duration in the subepi- when compared with the sub-
endocardium. Thus, in human ventricular myocardium the
change in MHC isoform composition during heart failure
will be variable and this may have obscured significant
effects on sarcomeric function. However, recent studies
have shown that even a small shift will have a significant
impact on cardiomyocyte contractility.
11,27
In contrast to
ventricular human tissue, human atria contain 80% of
a-MHC.
12,24
In atrial fibrillation, the b-MHC expression
increased almost two-fold, which coincided with a reduction
in kinetics of force re-development.
28
Overall, the shift towards the slow and more economical
b-MHC isoform occurs in human diseased atria and to a
lesser extent in diseased human ventricular myocardium.
The MHC shift may be beneficial under pathological con-
ditions, since less energy is required to maintain cardiac
pump function at rest, though at the expense of reduced
speed of contraction and power output.
2.2 Myosin light chains
Apart from the shift in MHC, changes may occur in the
expression pattern of MLCs within the heart. In particular,
isoform changes have been reported for MLC-1 (or essential
MLC), both in atrial and ventricular tissue. MLC-1 not only
interacts with MHC, but also binds to actin with its N-
terminus. It exists in two forms, a ventricular (VLC-1) and
an atrial (ALC-1) form.
29
The latter is expressed in the
entire heart muscle during foetal and early life and is sub-
sequently replaced by the ventricular form. In the ventricles
of patients with hypertrophic obstructive cardiomyopathy
relatively high amounts of ALC-1 were found, which corre-
lated with the maximal rate of force development.
30
Apart
from its positive effect on dynamics of contraction, replace-
ment of VLC-1 by ALC-1 increased isometric force develop-
ment of ventricular preparations at maximal and
submaximal activation.
29
Morano et al.
31
hypothesized that
MLC-1 tethers MHC to actin and thereby restrains cross-
bridge cycling and reduces force generation. As actin-
binding affinity of ALC-1 is less than of VLC-1, improved
cardiac function in ventricular tissue expressing ALC-1 may
be explained by weakening of the interaction between
actin and MLC-1. However, although up-regulation of ALC-1
may represent a compensatory mechanism to improve
cardiac function, it is not a consistent protein alteration in
ischaemic and idiopathic cardiomyopathy (IDCM), as large
individual differences exist.
29,30,32
Alternatively, addition of the N-terminal region of MLC-1
may be used to promote sarcomeric function. Cardiac func-
tion may be enhanced by disruption of the actin
–
MLC-1
interaction by additional expression of N-terminal MLC-1
fragments, which competitively bind to actin.
29,31
Another
explanation for MLC-1 induced increase in function was
given by Rarick et al.
33
who reported increased myofibrillar
MgATPase activity at submaximal [Ca
2þ
] upon addition of an
N-terminal VLC-1 fragment. These authors suggested that
the MLC-1 N-terminal peptide directly affects protein inter-
actions and exerts an inotropic effect via cooperative mech-
anisms, which activate the entire thin filament. Hence, the
N-terminus of MLC-1 seems to exert a beneficial effect on
sarcomeric function as increased systolic force generation
and rates of contraction and relaxation were observed in
Figure 1 Schematic representation of the cardiac sarcomere during diastole
(A) and systole (B). (A) The thin filament is composed of actin, tropomyosin,
and the troponin complex (composed of troponin T, cTnT; troponin C, cTnC;
and troponin I, cTnI). The thick filament is composed of myosin, an asym-
metric dimer composed of a globular head portion (S1), a hinged stalk
region (S2), and a rod section. The S1 portion of myosin contains both the
ATP hydrolysis domain and the actin-binding domain. Each myosin head is
associated with a pair of myosin light chains, which consists of an essential
light chain (MLC-1) and a regulatory light chain (MLC-2). MyBP-C, myosin
binding protein C. (B) Calcium binding to cTnC induces a conformational
rearrangement in the troponin
–
tropomyosin complex. Movement of tropo-
myosin exposes a myosin-binding site on actin allowing cross-bridge formation
to take place. This results in force development and/or shortening of the
sarcomere (Modified from de Tombe, J Biomech 2003;36:721
–
730, with
permission).
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hearts from transgenic rats harbouring minigenes encoding
the N-terminal domain of MLC-1.
34
Thus, although endogen-
ous heterogeneous expression of MLCs may be of minor rel-
evance in failing human myocardium, up-regulation of the
MLC-1 N-terminal fragment might provide a therapeutic
tool to enhance cardiac performance.
2.3 Troponin T
Anderson et al.
35
have proposed that re-expression of foetal
troponin T (cTnT) may also contribute to the reduced ATPase
activity in human heart failure. A significant inverse nega-
tive relationship was found between (re-)expression of
foetal cTnTand myofibrillar ATPase activity in human ventri-
cular tissue from normal and end-stage failing hearts.
However, this observation has not been confirmed by
others, and similar to the expression of a-MHC, the
expression levels of foetal cTnT is variable among individ-
uals.
36
–
38
While Anderson et al.
35
observed re-expression
of foetal cTnT in all end-stage failing human hearts, Solaro
et al.
36
only observed this foetal cTnT isoform in one out
of 10 failing heart samples. Similarly, we have observed
foetal cTnT in only one out of 24 patients with end-stage
heart failure.
38
Mesnard-Rouiller et al.
37
found expression
of foetal cTnT in half of the failing ventricles and suggested
that re-expression of foetal cTnT isoforms is not a common
characteristic of heart failure and most likely depends on
other factors such as intensity and duration of the elevation
of wall stress.
The consequences of foetal cTnT on sarcomeric contrac-
tion were studied upon exchange of endogenous cTnT with
foetal cTnT in rat cardiomyocytes.
39
No differences were
found in myofilament force development at maximal and
submaximal Ca
2þ
concentrations at a sarcomere length of
2.2 mm between cardiomyocytes exchanged with troponin
complex containing adult or foetal cTnT.
39
Akella et al.
40
observed a decrease in Ca
2þ
-responsiveness at low
(1.9 mm), but not at high (2.4 mm) sarcomere length in
skinned cardiac trabeculae from diabetic rats which
coincided with alterations in cTnT composition. More
recently, it was shown by Gomes et al.
41
that foetal
cTnT modulates Ca
2þ
sensitivity in the presence of foetal
(skeletal) TnI. Hence, the effect of foetal cTnT on sarco-
meric function seems to be dependent on sarcomere
length and protein background in the heart.
In conclusion, most isoform changes might be an intrinsic
part of the so-called ‘fetal’ (hypertrophic) program yet their
expression appears to be highly variable within human ven-
tricular tissue. Minor shifts in MHC and MLC composition do
not explain sarcomeric dysfunction in heart failure, but
are of compensatory nature.
3. Proteolysis and sarcomeric dysfunction
3.1 Troponin I
There is ample evidence that proteolytic activity is
enhanced after an acute ischaemic insult. Degradation pro-
ducts of several sarcomeric proteins
42
–
47
have been
observed, which may subsequently induce sarcomeric
dysfunction. One of the main proteins thought to be res-
ponsible for impaired cardiac function upon ischaemia/
reperfusion is cardiac troponin I (cTnI) (Figure 1).
42
–
45
In
rodents, McDonough et al.
44
showed that with moderate
ischaemia/reperfusion, cTnI is cleaved at its C-terminus,
which results in a truncated cTnI product (cTnI
1
–
193
,in
human cTnI
1
–
192
). More recently, it was postulated that
degradation of cTnI might also impair cardiomyocyte func-
tion and contribute to reduced pump function in heart
failure.
48
Myocardial infarction in pigs induced a reduction
in the maximal force generating capacity of single permea-
bilized cardiomyocytes isolated from remodelled non-
infarcted left ventricular tissue, in which minor degradation
of cTnI (4%) was observed.
49
In addition, independent of
ischaemia, cTnI degradation has been demonstrated in
human myocardium from coronary artery disease patients
with different degrees of heart failure.
45,50,51
To investigate
if truncated cTnI may contribute to depressed cardiac pump
function in human ischaemic cardiomyopathy and heart
failure, we recently investigated the direct functional
effects of cTnI
1
–
192
in human cardiomyocytes. Force
measurements were performed in non-failing human cardio-
myocytes permeabilized with Triton-X 100 and exchanged
with troponin complex containing either full length
(cTnI
FL
) or truncated cTnI.
52
Surprisingly, truncated cTnI
did not significantly alter maximal force development
(Figure 2A). Likewise, passive force was not different
between cells containing cTnI
FL
and cTnI
1
–
192
. However,
myofilament Ca
2þ
-sensitivity was significantly higher in
cTnI
1
–
192
exchanged preparations compared with cTnI
FL
cells (Figure 2B). This implicates that in humans truncation
of cTnI may limit relaxation of the heart muscle, while sys-
tolic function would benefit from the increase in Ca
2þ
-
responsiveness of the myofilaments.
4. Role of protein phosphorylation in
sarcomeric dysfunction
4.1 Protein kinase A-mediated phosphorylation
Not only truncation of cTnI, but also its phosphorylation
status is a prominent determinant of sarcomeric function,
both in health and disease. Upon b-adrenergic stimulation,
protein kinase A (PKA)-mediated cTnI phosphorylation at
serines 23/24 is associated with a decrease in myofilament
Ca
2þ
sensitivity
32,53,54
and contributes to an acceleration
of cardiac relaxation.
55,56
Since b-adrenergic signalling is
reduced in heart failure due to down-regulation and desen-
sitization of b-adrenoceptors,
57
–
59
PKA-mediated cTnI phos-
phorylation might be less pronounced in failing myocardium.
In agreement with reduced b-adrenergic signalling, reduced
phosphorylation levels of cTnI have been reported in failing
human myocardium compared with non-failing donor
hearts.
60
–
62
More specifically, phosphorylation at PKA sites
23/24 was significantly reduced in end-stage failing com-
pared with donor human myocardium.
61,62
At the functional
level, reduced PKA-mediated cTnI phosphorylation would
result in an increase in Ca
2þ
sensitivity of the myofilaments,
as was observed in single permeabilized human cardiomyo-
cytes isolated from end-stage failing hearts.
32,54,61,63
In
comparison with cells from non-failing donor myocardium,
Ca
2þ
sensitivity was significantly higher in cardiomyocytes
from patients with idiopathic or ischaemic cardiomyopathy
(Figure 3A). This sarcomeric defect was normalized upon
treatment of cardiomyocytes with the catalytic subunit of
PKA (Figure 3B), as the reduction in pCa
50
was larger in car-
diomyocytes from failing compared with donor hearts.
32
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Recently, Messer et al.
62
have shown that altered cTnI phos-
phorylation most likely underlies the increased Ca
2þ
-
responsiveness as isolated thin filaments from failing
human hearts displayed higher Ca
2þ
-responsiveness com-
pared with filaments from donor myocardium in an in vitro
motility assay. One should be careful when using non-failing
donor myocardium as ‘normal’, because of the high blood
catecholamine levels at the time of tissue procurement.
The high level of cTnI phosphorylation and relatively small
effect of PKA on myofilament Ca
2þ
-responsiveness might
reflect over-stimulation of the b-adrenergic pathway in
donors and thereby augment the difference between
healthy and failing samples. However, a similar increase
in myofilament Ca
2þ
-responsiveness has been observed in
several animal models of heart failure.
49,64
–
66
To minimize variable receptor stimulation at the time of
biopsy procurement, we recently conducted a series of
experiments on single cardiomyocytes from pigs with a myo-
cardial infarction or sham-operated animals isolated from
transmural needle biopsies, which were instantly frozen in
liquid nitrogen. Biopsies were taken from remote left ventri-
cular tissue 3 weeks after myocardial infarction induced by
ligation of the left circumflex coronary artery or from
sham-operated animals. Consistent with previous obser-
vations (Figure 4),
49
Ca
2þ
-responsiveness was significantly
higher in cells from infarct compared with sham animals,
while the shift upon PKA was smaller in sham than in post-
infarct remodelled myocardium (unpublished data). These
data clearly show that alterations in b-adrenergic signalling
and the concomitant reduction in PKA-mediated cTnI phos-
phorylation impair sarcomeric function. The increased
Ca
2þ
sensitivity of the myofilaments might contribute to
diastolic dysfunction via impaired relaxation of failing
myocardium.
4.2 Protein kinase C and D
Apart from the b-adrenergic pathway, other signalling
routes might be involved in the alterations in phosphoryl-
ation and function of sarcomeric proteins in heart failure.
Figure 2 Isometric force was measured in single permeabilized human cardiomyocytes upon activation in solutions containing maximal (pCa 4.5) and submax-
imal Ca
2þ
concentrations (pCa 5 to 6) and in relaxing solution (pCa 9) to determine maximal (F
max
) and passive (F
pas
) force development (A) and Ca
2þ
sensitivity of
force development (B). Force at submaximal activation was normalized to the force obtained during maximal activation. (A) The maximal force generating
capacity (F
max
) and F
pas
did not differ between cardiomyocytes containing truncated cTnI (cTn
1
–
192
) or full-length cTnI (cTn
FL
). (B)Ca
2þ
sensitivity increased
in cTn
1
–
192
cardiomyocytes compared with cTn
FL
exchanged cells (From Narolska et al.Circ Res 2006;99:1012
–
1020: Figures printed with permission).
Figure 3 (A)Ca
2þ
sensitivity was significantly higher in cardiomyocytes isolated from end-stage failing (n¼10) compared with donor (n¼6) hearts. (B) Incu-
bation of cells with the catalytic subunit of PKA reduced pCa
50
(DpCa
50
: 0.20 +0.01 and 0.03 +0.01 in failing and donor cells, respectively) and abolished the
difference between both groups (Modified from van der Velden et al.Cardiovasc Res 2003;57:37
–
47, with permission).
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Noteworthy, overall phosphorylation status of cTnI, deter-
mined on ProQ Diamond stained gels,
67
did not significantly
differ between sham-operated and MI pigs (unpublished
data), while the shift in Ca
2þ
sensitivity upon PKA treatment
was larger in infarct compared with sham animals (Figure
4B). This implies that, whereas PKA-mediated cTnI phos-
phorylation is down-regulated in infarct animals, cTnI phos-
phorylation by other kinases should be increased. One of the
most likely candidates is protein kinase C (PKC). Its activity
and expression levels are increased in heart failure
66,68
–
71
and both cTnI and cTnT contain specific PKC phosphorylata-
ble sites (i.e. serine 43/45 and threonine 144 in cTnI and
serine 201 and threonine 197/206/287 in cTnT).
72
–
74
In
addition to PKC, an increase in protein kinase D (PKD) has
been reported in heart failure.
75
PKD can be activated
upon phosphorylation by PKC, and thus may act down-
stream of PKC, or it also may be directly activated upon
receptor stimulation by, e.g. endothelin 1.
76
It has been
shown that PKD is able to reduce myofilament Ca
2þ
sensi-
tivity via phosphorylation of PKA-sites (serine 23/24) in
cTnI.
77
Consequently, the regulatory window of PKC and
PKD in sarcomeric function might be widened in diseased
myocardium.
The functional consequences of PKC-mediated protein
phosphorylation have been investigated in rodent models
and indicated a central role for cTnI and cTnT in reducing
maximal myofilament force development.
73,74,78
Apart
from its effect on maximal force, PKC has been shown to
reduce myofilament Ca
2þ
-sensitivity in rodent and human
myocardium.
61,71,73,74,78
The possible involvement of
PKC-mediated protein phosphorylation in sarcomeric func-
tion in failing myocardium was shown recently in rat
models of end-stage heart failure resulting from chronic
pressure overload (aortic banding) and myocardial infarc-
tion.
71
In both models increased expression and activation
of PKCawere observed in the late, but not in the early
phase of heart failure. Maximal force generating capacity
and Ca
2þ
-sensitivity of permeabilized cardiomyocytes were
significantly reduced in end-stage failing animals compared
with age-matched controls,
71,79
and both parameters
increased upon treatment with protein phosphatase 1
(PP-1). In contrast, PKCadid not significantly alter cardio-
myocyte function of failing cardiomyocytes, while it
reduced both maximal force and its Ca
2þ
sensitivity in
cells from the control group. In a previous study, the same
group performed experiments where in failing cardiomyo-
cytes the endogenous Tn-complex was exchanged by unpho-
sphorylated troponin complex, while control cells were
exchanged with troponin complex extracted from failing
hearts.
79
Upon exchange, Ca
2þ
sensitivity of failing cardio-
myocytes was restored towards the value observed in con-
trols, while failing troponin complex induced a significant
reduction in Ca
2þ
sensitivity in control cells. However, tro-
ponin exchange did not affect maximal tension, indicating
that PKC-mediated phosphorylation of troponin is not
involved in the reduced force generating capacity. Overall,
the data confirm that altered myofilament Ca
2þ
sensitivity
can be attributed to altered troponin phosphorylation,
while changes in maximal force generating capacity most
likely rely on the permissive action of other sarcomeric
proteins.
Whether PKC- and PKD-mediated phosphorylation and a
concurrent reduction in Ca
2þ
-responsiveness is detrimental
for cardiac function or represents an alternative mechanism
to preserve positive lusitropy during exercise and compen-
sates for reduced PKA-mediated cardiac relaxation requires
further investigation.
5. Sarcomeric dysfunction in heart failure
5.1 Increased vs. decreased Ca
21
sensitivity
Opposite to the increased myofilament Ca
2þ
sensitivity
observed in human end-stage failing myocardium
(Figure 3)
32,54,61,63
and in several animal models of cardiac
disease (Figure 4),
49,64
–
66
a decrease in myofilament Ca
2þ
-
sensitivity was reported in rodent models of heart failure
resulting from chronic pressure overload (aortic banding)
and myocardial infarction.
71,79
One possible explanation
for these contrasting observations might be the level of neu-
rohumoral stimulation present at the time of tissue procure-
ment. An intricate balance exists between kinase and
phosphatase activities within the cardiomyocyte as was
shown recently by Braz et al.
68
They reported that both
PKA and PKC may alter phosphorylation status of proteins
indirectly via phosphorylation of protein phosphatase
inhibitor-1 (I-1). Opposite to PKA, which suppresses PP-1
activity,
80
PKC enhances PP-1 activity via phosphorylation
of I-1. This illustrates the delicate balance between
kinases and phosphatases within a cell. An increase in PKC
Figure 4 Isometric force measurements were performed in single Triton-
permeabilized cardiomyocytes isolated from remote left ventricular tissue
from pigs 3 weeks after myocardial infarction. Maximal force development
(F
max
) was significantly lower in myocardial infarction compared with
sham-operated animals (A). Similar to the observations in human heart
failure, Ca
2þ
sensitivity was significantly higher (A) and the PKA-mediated
reduction in Ca
2þ
sensitivity was larger (B) in cells from myocardial infarction
compared with sham animals (Modified from van der Velden et al.Circ Res
2004;95:e85-e95, with permission).
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and a decrease in PKA-mediated phosphorylation of I-1
would enhance PP-1 activity
81
and thereby induce hypopho-
sphorylation of sarcomeric proteins.
Apart from differences in neurohumoral status when
tissue is retrieved from the heart, diverse alterations in
the signalling pathways known to alter sarcomeric protein
phosphorylation upon neurohumoral stimulation most likely
underlie diverse functional properties of the sarcomeres.
Already in 1991, Bristow et al.
82
have shown different
alterations in the b-adrenergic pathway in hearts with
ischaemic heart disease (ISHD) and IDCM. Analysis of sarco-
meric protein phosphorylation on ProQ Diamond stained
gradient gels (Figure 5A)
67
revealed significant differences
between left ventricular myocardial tissue from end-stage
failing patients with IDCM and ISHD (Figure 5B). Phosphoryl-
ation of cTnI was significantly higher in non-failing donor
compared with end-stage failing myocardium. In addition,
myosin binding protein C, which is phosphorylated upon
b-adrenergic stimulation is lower in failing compared with
donor hearts.
67,83
Noteworthy, MLC-2 phosphorylation was
significantly higher in ISHD compared with donor and IDCM
myocardium, and statistical analysis revealed significant
different phosphorylation of cTnI between ISHD and IDCM
samples. In line with a higher level of cTnI phosphorylation
in ISHD samples, myofilament Ca
2þ
sensitivity was signifi-
cantly lower in ISHD compared with IDCM myocardium.
32,54
These data provide evidence that diverse alterations in
sarcomeric protein composition and function in failing
hearts are related to underlying cause or phenotype.
5.2 Reduction in maximal force generating capacity
Reduced maximal force has been observed in diverse models
of cardiomyopathy (Figure 4A).
49,65,79,84
In rat with
pressure-overload and infarction-induced cardiomyopathy,
the reduction in F
max
amounted to 35 and 42%, respect-
ively.
79
As reduced F
max
was only partly reversed by PP-1
(15%),
71
and as described earlier, may not directly involve
the troponin complex,
79
alternative signalling routes, and
other sarcomeric proteins may be of relevance. Moreover,
depressed cardiomyocyte force development was also
observed in enzymatically isolated preparations from
failing rat hearts,
84
in which the isolation procedure most
likely reduced phosphorylation status of most sarcomeric
proteins. Therefore, part of the reduction in maximal
force might at least in part be related to altered isoform
composition and/or proteolysis of sarcomeric proteins. A
recent study in transgenic mice by Vahebi et al.,
85
in
which p38 MAPK (mitogen activated protein kinase) was
constitutively active in the heart, revealed a possible role
for tropomyosin dephosphorylation in the depression of
maximal force of the sarcomeres. Activation of p38 MAPK,
as occurs in pressure overload-induced hypertrophy, has
been shown to exert a negative effect on cardiomyocyte
contractility without altering Ca
2þ
-handling.
86
The study in
transgenic mice
85
indicated that apart from its role in remo-
delling and apoptosis, activated p38 MAPK leads to sarco-
meric dysfunction, possibly via activation of phosphatases
and a subsequent dephosphorylation of tropomyosin. The
level of tropomyosin phosphorylation appears to be species-
dependent, being relatively high in mice
85
and lower in
human myocardium (Figure 5A). However, similar to
isoform changes in MHC, small changes in phosphorylation
may exert a significant effect on sarcomeric function.
Therefore, the (patho)physiological role of tropomyosin
phosphorylation for sarcomeric function requires further
investigation.
In conclusion, depressed force development cannot be
explained by a single protein alteration, though seems to
be the result of complex interactions between various sarco-
meric proteins.
5.3 Increased cardiomyocyte stiffening
Subtle, though functionally important changes in protein
phosphorylation, induced by kinases and phosphatases
other than PKA, may have been obscured in failing human
myocardium in comparison with donor hearts. Separation
of patients with heart failure into subgroups, based on
severity, cause or phenotype, represents a powerful
approach to reveal the causes and functional implications
of alterations in sarcomeric function in human heart
failure. Comparison of patients with diastolic (DHF) and sys-
tolic heart failure (SHF) revealed an increased passive force
Figure 5 (A) Human heart samples (D, donor and IDCM, idiopathic dilated cardiomyopathy; 20 mg/lane) separated on 4
–
15% gradient gels. Phosphorylation of
myosin binding protein C (MyBP-C), desmin, troponin T (cTnT), troponin I (cTnI), and myosin light chain 2 (MLC-2) was determined with Pro-Q Diamond stain. (B)
Bar graph to illustrate differences in sarcomeric protein phosphorylation between human donor hearts and failing myocardium (ISHD, ischaemic heart failure).
*P,0.05 in one-way ANOVA. n, number of hearts [Modified from Zaremba et al.Proteomics Clin Appl 2007;1:1285
–
1290, with permission].
N. Hamdani et al.654
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development in cardiomyocytes from DHF compared with
SHF patients (Figure 6).
87
A significant positive correlation
was present between in vivo left ventricular end-diastolic
pressure (LVEDP) and F
pas
,
88
indicating that cardiomyocyte
stiffening contributes to high filling pressures in DHF.
Increased cardiomyocyte passive force was corrected to
values observed in hearts with preserved ejection fraction
and normal LVEDP
88
upon incubation with PKA (Figure 6),
indicative for hypophosphorylation of sarcomeric proteins.
The hypophosphorylated sarcomeric protein, possibly
titin,
89,90
could be a specific myocardial target for drug
therapy to lower LVEDP in DHF.
6. Future perspectives
The question if depressed cardiomyocyte contractility is
involved in heart failure has been positively answered.
Overall, there seems to be general consensus that sarco-
meric dysfunction in heart failure results from altered
protein phosphorylation, which is the result of complex
changes in kinase and phosphatase expression and activity.
The balance between kinases and phosphatases in the cardi-
omyocyte are humoral and heart rate-dependent and as a
consequence the activities of kinases and phosphatases
vary in time. Apart from temporal changes, spatial
changes occur, as complex interactions have been shown
between kinases and phosphatases regulating calcium
homeostasis within cardiomyocytes.
91,92
Such complex sig-
nalling may also apply to the myofilaments. Moreover,
within the complex pattern of sarcomeric protein phos-
phorylation each protein and its phosphorylation status
influences the behaviour of other sarcomeric proteins.
93,94
As sarcomeric function most likely reflects differences in
phosphorylation status and depend on the neurohumoral
status and heart rate at the time of tissue procurement,
investigation of the functional properties of the sarcomeres
should be performed in cardiac tissue, which is obtained
under standardized conditions. The use of catheter biopsies
has proved to be a major leap forward in unravelling
sarcomeric dysfunction in human myocardium.
87,88
Linkage
of in vivo haemodynamic data with cardiomyocyte force
measurements revealed that stiffening of the sarcomeres
contributes to impaired filling of the heart in DHF patients.
88
To obtain insight in dynamic signalling cardiac samples could
be retrieved upon receptor stimulation.
67
This approach
allows determination of the direct relation between func-
tional, structural, and protein characteristics at the cellular
level with in vivo haemodynamics measured at the time of
tissue harvesting.
The sarcomeric proteome will be even more complex than
described in the present review, since other signalling
routes, apart from the b-adrenergic pathway, may be trig-
gered under pathological conditions and affect sarcomeric
function.
68,75,76,86,95,96
Only recently, Yuan et al.
97
discov-
ered novel phosphorylation sites within the N-terminus of
MyBP-C, which were differentially phosphorylated upon
stunning in canine and rat myocardium. Apart from phos-
phorylation, post-translational modifications resulting from
oxidative stress might impair sarcomeric function. Moreover,
mutant sarcomeric proteins as found in inherited cardiomyo-
pathies
98,99
further complicate analysis of causality between
protein alterations and function of the sarcomere. Over the
past years knowledge on mutated sarcomeric proteins
present in cardiomyopathies increased swiftly. However,
the exact consequences of these mutations on cardiomyo-
cyte function in human cardiac tissue are still unclear and
knowledge concerning additional (mal)adaptive changes in
sarcomeric proteins is scarce. Hence, it remains to be eluci-
dated if and to what extent altered sarcomeric protein
expression and/or post-translational changes impair sarco-
meric function in inherited cardiomyopathies.
The combination of sarcomeric force measurements with
proteomic analysis (i.e. functional proteomics) will reveal
(novel) post-translational modifications involved in cardio-
myocyte dysfunction in heart failure. The use of transgenic
animal models and protein exchange experiments in
cardiac preparations are required to define the specific
role of post-translational protein modifications and mutant
sarcomeric proteins in cardiac function.
7. Clinical implications
The recently obtained data in human myocardium (Figures 5
and 6) indicate that divergent disturbance of receptor-
signalling cascades depend on underlying cause and pheno-
type. Diverse alterations in signalling pathways might alter
the responsiveness of patients to drug therapy and therefore
the current strategy of treating heart failure should be
re-evaluated. Large randomized, double-blind, placebo-
controlled multicentre trials have shown that treatment of
heart failure patients (classified according to the New York
Heart Association into class II
–
IV) with neurohumoral recep-
tor blockers, such as ACE-inhibitors and beta-blockers,
reduces both morbidity and mortality. However, it remains
to be investigated if and to what extent reversal of sarco-
meric dysfunction contributes to the beneficial effects of
beta-blocker and ACE-inhibitor therapy in different patient
groups.
Interestingly, exercise in mice with a myocardial infarc-
tion reversed depressed sarcomeric function to values
observed in controls.
65
The beneficial effects appeared to
be the result of improved b-adrenergic signalling. Future
studies should investigate if the combination of currently
used neurohumoral blockers with exercise yield added
benefit. Novel therapy may include drugs targeted to
mediators down-stream of the adrenergic and angiotensin
receptors. Cardiac performance may be improved by target-
ing a specific myofilament protein
34,100
to directly modulate
Figure 6 Cardiomyocyte stiffening in human heart failure. Cardiomyocyte
passive force (F
pas
) was significantly higher in DHF than in SHF (*P,0.05).
PKA treatment reduced F
pas
in SHF and DHF (
#
P,0.05 in paired t-test)
(From van Heerebeek et al. Circulation 2006;113:1966
–
1973, with
permission).
Sarcomeric dysfunction in heart failure 655
by guest on June 5, 2013http://cardiovascres.oxfordjournals.org/Downloaded from
sarcomeric function. Further exploration of the complex sig-
nalling routes underlying defects in sarcomeric function is
required in order to develop more precise, individualized
therapy in heart failure patients.
Conflict of interest: none declared.
Funding
Netherlands Organisation for Scientific Research (VENI grant
916.36.013 to J.v.d.V.); Netherlands Heart Foundation
(grant 2000T042 to D.J.D., grant 2005B220 to J.v.d.V.).
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