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Cardiovascular Research 59 (2003) 668–677
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Altered force–frequency response in non-failing hearts with decreased
SERCA pump-level
Sabine Huke a , Lynne H. Liu a , Danuta Biniakiewicz b , William T. Abraham b ,
Muthu Periasamy a , *
a
Department of Physiology and Cell Biology, Ohio State University College of Medicine and Public Health, 304 Hamilton Hall, 1645 Neil Avenue,
Columbus, OH 43210 -1218, USA
b
Division of Cardiovascular Medicine, Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA
Received 4 February 2003; received in revised form 30 April 2003; accepted 7 May 2003
Abstract
Objective: Decreased SERCA2 activity is considered to significantly contribute to the contractile dysfunction of failing hearts.
However, it is now known how decreases in SERCA activity affect cardiac function in detail and also if a decrease alone is sufficient to
cause heart failure. Methods: SERCA2 (1 / 2) gene-targeted mice (HET) were generated and heart function was analyzed using the
isolated work-performing heart technique. Plasma and cardiac catecholamine levels were determined at three, six and nine months of age
and heart sections from twelve months old mice subjected to standard histological analysis. Results: We demonstrate that reduced
expression of SERCA does not lead to cardiac hypertrophy or fibrosis and does not increase resting plasma-norepinephrine levels in HET
mice. However, isolated perfused HET hearts exhibited decreased maximal rates of contraction and relaxation and prolonged
time-parameters. The ability of the HET hearts to respond to increases in load (Starling) was not affected and they responded
appropriately to b-adrenergic stimulation. In contrast, the positive force-frequency response found in control hearts was not observed in
the HET hearts. The response was flat and three out of five HET hearts failed to maintain work at 550 beats / min. Conclusions: We
conclude that the SERCA2 pump level is a critical positive determinant of cardiac contractility and force-frequency relation.
 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Ablation; Ca-pump; Calcium; Contractile function; Ventricular function
1. Introduction
The sarcoplasmic reticulum (SR) Ca 21 ATPase
(SERCA) in the heart re-sequesters the Ca 21 released into
the cytosol during each contraction thereby fulfilling two
important functions: (1) by lowering the cytosolic Ca 21
concentration it promotes muscle relaxation and (2) by
replenishing the intracellular Ca 21 stores it provides Ca 21
needed for the next contraction (see also Ref. [1]).
Decreases in SERCA pump expression and activity were
observed in a variety of pathological conditions. Varying
degrees of defects in the SR Ca 21 uptake function have
been identified in animal models of heart disease and have
*Corresponding author. Tel.: 11-614-292-2310; fax: 11-614-2924888.
E-mail address: [email protected] (M. Periasamy).
been shown to correlate with altered contractile function.
Studies from many laboratories have shown that the
expression level of SERCA is significantly decreased in
animal models of pressure overload (PO)-induced hypertrophy / heart failure [2–6]. In cardiac tissue from these
and other PO models decreased SR calcium transport and
formation of the phospho-enzyme intermediate, E-P, was
observed [3–5,7,8].
In addition to studies using animal models of cardiac
diseases, there are considerable data indicating that SR
Ca 21 transport function is altered in end-stage human heart
failure [7,9,10]. A decrease in the level of SR Ca 21
ATPase (mRNA or protein or activity) was closely correlated with decreased myocardial function and impaired
force–frequency response [11–15].
Time for primary review 26 days.
0008-6363 / 03 / $ – see front matter  2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016 / S0008-6363(03)00436-X
S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
These studies suggest that SERCA pump level is critical
for the maintenance of cardiac function, but the true
relationship between SERCA levels and muscle function
cannot be defined. Interpretation of studies using tissues
from end-stage failing hearts is difficult because multiple
changes occur within myocytes during the progression into
heart failure. Therefore, a clear understanding of the
significance of SERCA pump expression and activity for
heart function has not yet been established.
To better define whether reduced SERCA levels affect
cardiac function and potentially result in heart failure we
used gene-targeting techniques to generate a mouse model
with decreased SERCA levels in the heart [16,17]. As
expected, the disruption of both copies of SERCA2 gene is
lethal, whereas heterozygous mice (HET) with a single
functional allele are alive and reproduce well. These mice
provide us with an in vivo model system to study how a
decrease in SERCA pump levels affect the contraction–
relaxation cycle of the heart. HET mice showed decreased
SERCA2a protein levels as well as a lower maximal
velocity of SR Ca 21 uptake, both reduced by about 35%
[16,17]. Analysis of in vivo cardiac function using a Millar
catheter revealed decreased maximal rates of contraction
and relaxation and lower mean arterial pressure in HET
mice. However, the heart weight to body weight ratio was
normal in HET mice. All these earlier studies were done in
young adult mice.
Several important points still need to be addressed: (1)
whether the reduction in SERCA pump expression causes
cardiac pathology as the mice age; (2) if the sympathetic
nervous system is activated in the mutant mice in order to
stimulate the heart and (3) how a decrease in SERCA level
modifies the ability of the heart to respond to increased
physiological demands, in particular to increases in load
(Frank–Starling response), b-adrenergic stimulation and
increases in heart rate (force–frequency response).
669
initially plus a second dose of 8 ml / g BW after 20 min).
Plasma and tissue levels were measured using a highperformance liquid chromatographic pump (Model 582)
attached to a HR-80 column with a mobile phase (Cat-APhase II) at a flow rate of 1.2 ml / min (equipment from
ESA, Chelmsford, MA, USA).
2.3. Isolated heart perfusions (‘ work-performing heart’)
The experimental conditions for the work-performing
mouse heart preparations were described previously [19].
Briefly, the hearts were attached by the aorta to a 20-gauge
cannula and temporarily retrogradely perfused with oxygenized modified Krebs–Henseleit solution. For the measurement of intraventricular pressure, a PE-50 catheter was
inserted into the apex of the left ventricle. The pulmonary
vein was connected to a second cannula and antegrade
perfusion was initiated with a basal workload of 250
mmHg ml / min (5 ml / min venous return and 50 mmHg
mean aortic pressure). Hearts were allowed to equilibrate
for 30 min before treatment.
Changes in afterload were accomplished by sequential
increases and decreases of aortic pressure in increments of
10 mmHg from 40 to 80 mmHg. A cumulative isoproterenol dose–response curve was obtained by
ISOPREL  (Abbot, Chicago, IL, USA; 0.2 mg / ml) infusion from 0.8 to 40 nmol / l, while each dose was applied
for 5 min. To measure the force–frequency response,
hearts were paced through electrodes connected to the
aortic and venous return cannulas using a Grass S48
stimulator. The hearts were stimulated up to 550 beats / min
in increments of 50 beats / min. Baseline loading conditions
were maintained during all experiments
(except where the
21
loading was varied). The free Ca concentration of the
perfusion buffer was generally 1.96 mmol / l, while it was
reduced to 1.45 mmol / l in the pacing experiments.
2.4. Statistical analysis
2. Methods
2.1. Transgenic mice
The generation of SERCA2 targeted mice was described
previously [16]. Littermate pairs (11–15 weeks old) of a
mixed background (129 / SvJ, Black Swiss) and of either
sex were used for all experiments unless otherwise noted.
The investigation conforms with the Guide for the Care
and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication no. 85-23,
revised 1996).
Results were expressed as mean6standard error of the
mean (S.E.M.). Significance was estimated by Student’s
t-test for paired and unpaired observations and one-way
repeated measures analysis of variance (ANOVA) followed
by a t-test for comparison of pair of points as appropriate
(SPSS for Windows 11.0). Regression lines were compared via testing for a difference in slope and elevation
(GraphPad Prism 3.0). A P#0.05 was considered significant.
3. Results
2.2. Tissue and plasma catecholamine levels
3.1. SERCA2 HET hearts do not develop hypertrophy
Catecholamine levels were determined essentially as
described elsewhere [18]. Briefly, blood and heart were
collected following prolonged anesthesia for 45 min with
Avertin i.p. (2.5% tribromoethanol; 16–18 ml / g BW
We described earlier that there is no evidence for cardiac
hypertrophy in young mutant mice, as indicated by an
unchanged heart weight to body weight ratio [16]. To
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S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
determine whether a reduction in SERCA pump level is
sufficient to induce heart disease in older animals, sexmatched heterozygous and control siblings (WT) were
aged. At 6 and 9 months of age the heart-to-body weight
ratio was unchanged in HET mice compared to control (6
months: 4.7260.16 in WT (n59) vs. 4.7360.21 in HET
(n57); 9 months: 4.9760.14 in WT (n518) vs. 5.0260.19
in HET (n518)). Six pairs of mice were aged up to 12
months and hearts were subjected to histological analysis
via conventional microscopic evaluation of hematoxylin /
eosin and Masson’s trichrome stained slices (Fig. 1).
Tissue morphology (structure, myofibrillar organization)
appeared normal in all hearts and there was no apparent
increase in interstitial fibrotic tissue content when HET
hearts were compared to their littermate controls. However, one female HET mouse showed mild cardiac dilation
and aortic thrombus formation, which is occasionally
found in older mice.
Overall, mutant mice do not display morphologic abnormalities indicative of heart failure up to 1 year of age.
3.2. Sympathetic activation is not increased in SERCA2
HET mice
One of the earliest events during progression into heart
failure is the activation of hormonal systems that modulate
both vascular tone and the retention of salt and water, e.g.
the sympathetic nervous system. Increased sympathetic
nerve activity can be demonstrated by increased plasma
norepinephrine concentrations and / or depleted myocardial
norepinephrine stores [20]. It has been shown that increased plasma norepinephrine levels precede the development of heart failure symptoms in patients [21] and,
therefore, likely precede cellular or structural changes of
the myocardium.
To determine if the sympathetic nervous system is
activated in HET mice we determined resting plasma
norepinephrine (NE) levels and cardiac NE content at the
age of 3, 6 and 9 months (Fig. 2). HET mice do not show
increased NE plasma levels or depleted cardiac NE stores.
Additionally, plasma epinephrine or dopamine levels were
unchanged between groups at all ages. Thus, there is no
evidence for a general or local cardiac sympathetic activation in these mice.
When the results were analyzed according to gender,
3-month-old female mice showed a decrease in cardiac
tissue NE level (1170666 ng / g HW in WT (n58) vs.
923645 ng / g HW in HET (n58)). This decrease was not
accompanied by increased plasma NE levels and was not
evident in older females.
Fig. 1. Representative slices of heart tissue stained with hematoxylin / eosin (A, B) and Masson’s trichrome (C, D). Four 1-year-old littermate pairs
(wild-type (WT) and SERCA2 heterozygous (HET)) of both sexes were studied. The magnification of the objective used is indicated on the right.
S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
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Fig. 2. Plasma (A) and heart tissue (B) norepinephrine (NE) levels in 3, 6 and 9 month old WT and HET mice. There are no statistical differences between
groups within each age set. Note that plasma NE levels decrease with age in the WT.
Moreover, we made two additional important observations related to age and gender in WT mice. Firstly, the
tissue NE levels remain steady between 3 and 9 months of
age, but the plasma NE levels actually decrease with age in
WT mice (P50.03; see also Fig. 1). Secondly, we observed a gender difference in the tissue NE levels. Female
WT hearts from 3-month-old mice contained 1170666
ng / g HW (n58) while male WT hearts contained only
818630 ng / g HW (n58) (P50.0003). Plasma NE levels
between males and females were not different (P50.5).
However, in older mice this gender difference in tissue NE
levels was not observed (female 1104694 ng / g HW (n5
9) vs. male 873680 ng / g HW (n59) (P50.08)). None of
the age- and gender-related observations were significant
in HET mice, while following the same trend.
3.3. Baseline contractility in HET hearts is decreased
To analyze cardiac function in the absence of any
neurohormonal stimulation and under controlled loading
conditions we used the isolated work-performing heart
set-up. HET hearts showed a moderate but consistent
decrease in the maximal rates of contraction and relaxation
as well as an increase in the time to peak pressure (TPP;
normalized to the amplitude of the pressure increase) and
time to 50% relaxation (RT 1 / 2 ; normalized to the
amplitude / 2 of the pressure decline) (Fig. 3).
3.4. Normal response to loading and b -adrenergic
stimulation
Next we determined how the HET hearts respond to
acute functional stress. First we increased the loading of
the heart via increases in mean aortic pressure (MAP) and,
therefore, increased afterload. The capacity of the ventricle
to vary the force of contraction as a function of the load is
generally referred to as the Frank–Starling mechanism.
Both WT as well as HET hearts showed an increase in the
maximal rates of contraction and relaxation and a decrease
in time parameters with increasing MAP (Fig. 4). While
contractility parameters were decreased and time parameters were increased, the slope of the response was unaffected in HET hearts (P values ranging from 0.15 to 0.91).
Secondly, a cumulative dose–response to isoproterenol
(b 1 and b 2 -agonist) was obtained (0.08–40 nmol / l; Fig.
5). The activation of SERCA due to the phosphorylation of
phospholamban seems to be critical in the heart’s contractile response to b-adrenergic stimulation. However, not
much is known how changes in SERCA expression level
modify b-adrenergic effects on cardiac function. In the
HET hearts, the dose–response was fully maintained. The
maximal rates of contraction and relaxation increased
simultaneously and were not statistically different. The
time parameters, however, were prolonged in the HET
hearts at the beginning of the iso-treatment, but reached the
same level as the control hearts at higher doses of iso.
To test if the maximal response to iso is different in
HET hearts, the hearts were perfused with iso-concentrations higher than 40 nM, i.e. 80 and 160 nM. Not all
hearts tolerated higher doses at first, but sometimes
recovered after a longer infusion period. The maximal
contractility was determined for each heart (independent if
this occurred at 80 nmol / l or 160 nmol / l). The highest
maximal rates of contraction were similar in both groups
(70396313 mmHg / s (WT) and 63236173 mmHg / s
(HET); P50.106), as well as the time parameters. How-
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Fig. 3. Cardiac functional parameters obtained from WT and HET mice using the isolated work-performing mouse heart preparation. Data are derived from
10 littermate pairs. (A) 1dP/ dt max, maximal rate of contraction; (B) 2dP/ dt max, maximal rate of relaxation; (C) TPP, time to peak pressure normalized
to the pressure amplitude (systolic minus end-diastolic pressure); (D) RT 1 / 2 , time to 50% relaxation normalized to the pressure amplitude (systolic minus
diastolic pressure divided by two). Heart rate (308610.1 beats / min in WT vs. 29467.1 beats / min in HET), end-diastolic pressure (5.360.7 mmHg in WT
vs. 6.460.7 in HET), and left atrial pressure (5.260.3 mmHg in WT vs. 5.560.3 mmHg in HET) were not different in HET hearts (all n510).
ever, the corresponding highest maximal rates of relaxation
were decreased in the HET hearts (69406206 mmHg / s
(WT) vs. 62676138 mmHg / s (HET), P50.038).
When values were expressed as % of baseline values
(baseline5100%) the increase of the maximal rate of
relaxation in response to iso was higher in the HET hearts
(maximal rate of relaxation increased up to 18466% (WT)
vs. 20365% (HET) and time to 50% relaxation per mmHg
decreased to 47.861.8% (WT) and 40.861.4% (HET)).
In summary, while the overall contractility is slightly
depressed, HET hearts respond appropriately to loading
and stimulation with isoproterenol. Thus, these two mechanisms are fully maintained in the SERCA2 (HET) mice.
3.5. Altered force–frequency response in het hearts
The third mechanism we studied was the force–frequency response (Fig. 6). In most mammalian species,
including mice [22], a positive force–frequency relation
has been observed. The relation is called ‘positive’ when
contractility increases with higher stimulation frequencies.
In the control hearts contractility increased, with a maximum at 450 beats / min and then decreased again when
paced higher (typical optimum-curve). In contrast, the
response in the HET hearts was flat and two out of five
hearts were not able to perform the basal workload at 500
beats / min. One additional HET heart failed when stimulated at 550 beats / min, therefore only two hearts maintained function at that high heart rate. However, their
function was poor and their left atrial pressure was high, a
clear indication of a pre-failing heart. In conclusion, the
WT hearts show a ‘positive’ force–frequency response,
whereas the ability of the HET hearts to potentiate their
contractility in response to increases in heart rate is
impaired.
It is important to note that the HET hearts did not show
functional deficits when the heart rate was increased as a
result of iso infusion. The heart rate response to iso was
unchanged in the HET (P50.372), while it remained lower
S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
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Fig. 4. Cardiac functional parameters obtained from WT and HET hearts in response to changes in afterload. Changes in afterload were achieved varying
the mean aortic pressure (MAP) from 40 to 70 mmHg. Data are derived from five littermate pairs. (A) and (C) show parameters of contraction, (B) and (D)
parameters of relaxation.
than 500 beats / min in both groups (at 40 nmol / l iso:
473610 beats / min (WT) vs. 440613 beats / min (HET)).
4. Discussion
In congestive heart failure a decreased expression and
activity of SERCA pump has been observed. A decrease in
SERCA pump activity is often correlated with cardiac
dysfunction and heart failure. However, it is not known
whether decreases in SERCA expression affect cardiac
function and also if this defect alone is sufficient to cause
heart failure. We addressed these questions using genetargeted mice, whose primary defect is the loss of one
allele of SERCA2.
An important finding of this study is that SERCA2 HET
mice do not develop cardiac hypertrophy or show any
signs of cardiac pathology up to 1 year of age. Thus, the
reduced levels of SERCA itself does not cause structural
changes found in hypertrophied / failing hearts. However,
two points need to be considered: we reported previously
changes in the expression level of other Ca 21 handling
proteins in HET mice, a decrease in SERCA inhibitor
phospholamban (PLB) and an increase in the sarcolemmal
Na / Ca 21 exchanger (NCX; [17]). The decrease in the
SERCA-inhibitor PLB might lead to a higher activation of
the remaining SERCA pumps and the increased activity of
NCX might enhance cytosolic Ca 21 removal and help to
largely preserve cardiac relaxation. Thus, these adaptations
could compensate, at least in part, for the decrease in
SERCA pump. Secondly, HET mice tend to develop
squamous cell tumors of the skin and the upper digestive
tract after about 12 months of age, which is associated with
early mortality [23]. Thus, the effect of decreased SERCA
expression on cardiac pathology cannot be examined
throughout the normal mouse-lifespan of about 2 years. It
has been shown that high phospholamban overexpression
(fourfold PLB overexpression) leads to overt heart failure
at the age of 15 months, followed by an early mortality
between 15 and 18 months [24]. Thus, we cannot rule out
that we might observe a cardiac phenotype in HET mice
older than 12 months.
In the PLB-overexpression model mentioned above
heart failure was preceded by an increased adrenergic
drive, associated with increased phosphorylation of phospholamban. We have also shown that the phosphorylation
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S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
Fig. 5. Cumulative dose–response to isoproterenol (0.08–40 nmol / l) in isolated work-performing hearts. Data are derived from five WT and four HET
mice. (A) and (C) show parameters of contraction, (B) and (D) parameters of relaxation. Stars indicate statistical differences between groups.
of PLB is increased at both phosphorylation sites in HET
mice [17,25]. Therefore we tested if the decrease in
SERCA pump expression causes an increased adrenergic
drive in HET mice and measured plasma and tissue
norepinephrine (NE) levels. Increased plasma NE levels
and decreased cardiac tissue levels can both indicate
increased adrenergic stimulation. None of this was observed in HET mice.
The preserved response to iso additionally supports the
absence of a chronic b-adrenergic drive. Chronic b-stimulation, as observed in heart failure, leads to phosphorylation and uncoupling of b-receptors and subsequently to the
loss of responsiveness to b-agonists (for overview see Ref.
[26]). Therefore, the full response to the b-agonist iso in
HET hearts supports the view that HET hearts are not
chronically exposed to catecholamines in vivo.
In theory, the SERCA2 HET mice and the high PLB
overexpression mice are very similar models which both
exhibit decreased SERCA2 activity. However, why PLB
overexpression, but not partial SERCA pump loss, induces
heightened sympathetic tone is unclear. PLB decreases the
apparent Ca 21 affinity of the pump, but does not decrease
the maximal velocity of the Ca 21 uptake, while in contrast
a decrease in SERCA reduces the velocity but does not
alter the affinity. We can speculate that these two different
modifications affect cardiac function differently and therefore have different effects on the sympathoadrenergic
system.
Taken together, this leads to two important conclusions:
(1) the increased phosphorylation of PLB we found earlier
in HET mice is independent of increased neurohormonal
stimulation and therefore due to an inherent mechanism
which is not yet understood and (2) cardiac function in
vivo in HET mice is adequate and does not cause
activation of the sympathetic nervous system.
Another important goal of this study was to determine
how HET hearts respond to increased physiological stress
and demand. Using the isolated work-performing heart
set-up, we found that HET hearts showed a moderate
reduction in baseline cardiac contractility in a magnitude
similar to our in vivo functional studies [16].
An intrinsic property of the heart is its ability to vary
S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
675
Fig. 6. Functional parameters in response to increases in heart rate (up to 550 beats / min) in isolated work-performing mouse hearts. Data are derived from
four WT and five HET mice. Data are derived from five WT and four HET mice. (A) and (C) show parameters of contraction, (B) and (D) parameters of
relaxation. Stars indicate statistical differences between groups.
contractility as a function of preload, namely Frank–Starling response. This fundamental capacity of the heart is
largely based on the myocardial length–tension relationship. Changes in intracellular SR Ca 21 release have been
implicated as a potential mechanism contributing to the
increase in force in response to stretch [27–30]. Vahl et al.
[31] reported a flatter slope of the length–force relationship in human failing hearts, which they partly attributed
to altered intracellular Ca 21 handling in failing myocardium. However, it is still a controversial subject if the
Frank–Starling mechanism is indeed altered in heart
failure [32–34]. While this remains unclear, the present
study shows that a decrease in SERCA pump expression
does not affect the slope of the Frank–Starling response in
HET hearts.
A change in the expression level of SERCA might lead
to an altered dose–response to b-agonists. The activation
of SERCA due to phosphorylation of phospholamban is
considered to be a key event in the b-adrenergic signal
transduction, leading to increased contractility and faster
relaxation of the heart. However, the decreased levels of
SERCA did not affect the b-adrenergic response in HET
hearts.
We have previously reported that the expression of PLB
is decreased by about 40% in HET mice, similar to the
decrease in SERCA pump level [17]. Therefore, the
relative PLB-to-SERCA ratio, which has been shown to be
an important determinant of myocardial contractility [35–
39], remains unchanged in HET hearts. The maintained
ratio of pump and regulator could explain that HET hearts
respond normally to iso. It is still intriguing that HET
hearts show the same magnitude of response, reaching
similar maximal contractile values as the WT hearts.
Future studies will address if other mechanisms are altered
that might help to preserve the b-adrenergic response in
the HET, e.g. altered protein-kinase or phosphatase activities.
Finally, we studied the response of the hearts to
increases in stimulation frequency. In the mouse contractility normally rises with increases in heart rate. While we
observed this ‘positive’ response in the WT littermates, the
force–frequency response was severely impaired in the
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S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
HET mice. Cardiac contractility drastically decreased at
higher stimulation rates. Thus, the HET hearts show clear
functional deficits at high heart rates.
We can hypothesize that this is directly due to the
decrease in SR Ca 21 transport. It is likely that the
diminished capability of the SR to re-sequester calcium in
HET mice becomes more apparent at higher stimulation
rates, as the available time for calcium transport decreases.
The total SERCA expression rather than the PLB-toSERCA ratio appears to be a determinant of the force–
frequency in this model.
The HET hearts resemble human failing hearts, in which
the frequency potentiation of contractile performance is
also blunted or ‘negative’. It has been shown that the
altered force–frequency relation results at least in part
from disturbed calcium handling, with decreased instead of
increased calcium transients as the stimulation frequency
rises [14,40,41]. Several studies found a correlation between impairment in the force–frequency potentiation in
failing hearts and a decrease in SERCA pump activity
[42–44].
In the present study, for the first time, we show a clear
relationship between decreased SERCA pump expression
and altered force–frequency response in non-failing cardiac tissue. However, the interpretation has to take into
account the decrease in the expression level of PLB and
the increase in NCX expression. The expression level of
both proteins has been implicated to affect the force–
frequency response [44–47]. One of these studies describes a ‘negative’ force–frequency response in human
failing hearts with increased NCX, but unchanged SERCA
expression [44].
In summary, we demonstrate that a reduction in SERCA
pump expression (|35%) in the heart does not lead to
cardiac hypertrophy and failure in aged 12-month-old
mice. HET hearts have the intrinsic ability to maintain
sufficient heart function and do not require sympathetic
stimulation, as shown in this study by the absence of an
increased tonic b-adrenergic drive in HET mice. However,
when analyzed ex vivo cardiac contractility is moderately
reduced. The response to load and b-agonists is maintained, while the ability to potentiate the contractility in
response to an increase in heart rate is lost. However, a
‘positive’ force–frequency response does not appear to be
imperative for animal survival. Decreased basal contractility and an impaired force–frequency response may not
necessarily lead to apparent cardiac failure.
Acknowledgements
The authors wish to thank Dr. Ingrid L. Grupp for
excellent advice regarding the work-performing heart
experiments and Gilbert Newman for his expert technical
assistance. We also thank Dr. Donna F. Kusewitt for her
valuable help with the histological analysis. This work was
supported by grants from the NIH (HL64140-02) and
American Heart Association (AHA 9950570N). S.H. was
supported by post-doctoral fellowships from the Deutsche
Forschungsgemeinschaft (HU 898 / 1-1) and AHA
(0120149B).
References
[1] Periasamy M, Huke S. SERCA pump level is a critical determinant
of Ca(21) homeostasis and cardiac contractility. J Mol Cell Cardiol
2001;33:1053–1063.
[2] Nagai R, Herzberg AZ, Brandl CJ et al. Regulation of myocardial
Ca 21 ATPase and phospholamban mRNA expression in response to
pressure overload and thyroid hormone. Proc Natl Acad Sci USA
1989;86:2966–2970.
[3] Feldman AM, Weinberg EO, Ray PE, Lorell BH. Selective changes
in cardiac gene expression during compensated hypertrophy and the
transition to cardiac decompensation in rats with chronic aortic
banding. Circ Res 1993;73:184–192.
[4] Matsui H, MacLennan DH, Alpert N, Periasamy M. Sarcoplasmic
reticulum gene expression in pressure overload-induced cardiac
hypertrophy in rabbit. Am J Physiol 1995;268:C252–C258.
[5] Qi M, Shannon TR, Euler DE, Bers DM, Samarel AM. Downregulation of sarcoplasmic reticulum Ca 21 -ATPase during progression of
left ventricular hypertrophy. Am J Physiol 1997;272:H2416–H2424.
[6] Aoyagi T, Yonekura K, Eto Y et al. The sarcoplasmic reticulum
Ca 21 -ATPase (SERCA2) gene promoter activity is decreased in
response to severe left ventricular pressure-overload hypertrophy in
rat hearts. J Mol Cell Cardiol 1999;31:919–926.
[7] Arai M, Matsui H, Perisamy M. Sarcoendoplasmic reticulum gene
expression in cardiac hypertrophy and heart failure. Circ Res
1994;74:555–564.
[8] Kiss E, Ball NA, Kranias EG, Walsh RA. Differential changes in
cardiac phospholamban and sarcoplasmic reticular Ca(21)-ATPase
protein levels. Effects on Ca 21 transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure.
Circ Res 1995;77:759–764.
[9] Hasenfuss G, Reinecke H, Studer R et al. Calcium cycling proteins
and force–frequency relationship in heart failure. Basic Res Cardiol
1996;91(Suppl. 2):17–22.
[10] Hasenfuss G. Alteration of calcium-regulatory proteins in heart
failure. Cardiovasc Res 1998;37:279–289.
[11] Arai M, Alpert NR, MacLennan DH, Barton P, Periasamy M.
Alterations in sarcoplasmic reticulum gene expression in human
heart failure: a possible mechanism for alterations in systolic and
diastolic properties of the failing myocardium. Circ Res
1993;72:463–469.
[12] Hasenfuss G, Reinecke H, Studer R et al. Relation between
myocardial function and expression of sarcoplasmic reticulum Ca 21 ATPase in failing and nonfailing human myocardium. Circ Res
1994;75:434–442.
[13] Mercadier JJ, Lompre AM, Duc P et al. Altered sarcoplasmic
reticulum Ca(21)-ATPase gene expression in the human ventricle
during end-stage heart failure. J Clin Invest 1990;85:305–309.
[14] Pieske B, Kretschmann B, Meyer M et al. Alterations in intracellular
calcium handling associated with the inverse force–frequency
relation in human dilated cardiomyopathy. Circulation
1995;92:1169–1178.
[15] Bavendiek U, Brixius K, Munch G et al. Effect of inotropic
interventions on the force–frequency relation in the human heart.
Basic Res Cardiol 1998;93(Suppl. 1):76–85.
[16] Periasamy M, Reed TD, Liu LH et al. Impaired cardiac performance
in heterozygous mice with a null mutation in the sarco(endo)plasmic
S. Huke et al. / Cardiovascular Research 59 (2003) 668–677
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
reticulum Ca 21 -ATPase isoform 2 (SERCA2) gene. J Biol Chem
1999;274:2556–2562.
Ji Y, Lalli MJ, Babu GJ et al. Disruption of a single copy of the
SERCA2 gene results in altered Ca 21 homeostasis and cardiomyocyte function. J Biol Chem 2000;275:38073–38080.
De Saint Blanquat G, Lamboeuf Y, Fritsch P. Determination of
catecholamines in biological tissues by liquid chromatography with
coulometric detection. J Chromatogr 1987;415:388–392.
Grupp IL, Grupp G, Syfris G. The isolated work-performing mouse
heart preparation. Comparison and quantification of cardiac performance in transgenic and wild-type mice. In: Walsh RA, Hoid BD,
editors, Cardiovascular physiology in the genetically engineered
mouse, Boston, MA: Kluwer; 1998, pp. 77–95.
Brunner-La Rocca HP, Esler MD, Jennings GL, Kaye DM. Effect of
cardiac sympathetic nervous activity on mode of death in congestive
heart failure. Eur Heart J 2001;22:1136–1143.
Benedict CR, Shelton B, Johnstone DE et al. Prognostic significance
of plasma norepinephrine in patients with asymptomatic left ventricular
dysfunction,
SOLVD
Investigators.
Circulation
1996;15:690–697.
Gao WD, Perez NG, Marban E. Calcium cycling and contractile
activation in intact mouse cardiac muscle. J Physiol 1998;507:175–
184.
Liu LH, Boivin GP, Prasad V, Periasamy M, Shull GE. Squamous
cell tumors in mice heterozygous for a null allele of Atp2a2,
encoding the sarco(endo)plasmic reticulum Ca 21 -ATPase isoform 2
Ca 21 pump. J Biol Chem 2001;276:26737–26740.
Dash R, Kadambi FJ, Schmidt AG et al. Interactions between
phospholamban and b-adrenergic drive may lead to cardiomyopathy
and early mortality. Circulation 2001;103:889–896.
Ji Y, Netticadan T, Liu LH, Shull GE, Periasamy M. Decreased
cardiac SERCA2 calcium pump activity results in decreased phospholamban protein levels and increased compensatory phosphorylation at phospholamban residues Ser-16 and Thr-17. Circulation
2000;102(Suppl. S):1447, Abstract.
Hajjar RJ, Muller FU, Schmitz W, Schnabel P, Bohm M. Molecular
aspects of adrenergic signal transduction in cardiac failure. J Mol
Med 1998;76:747–755.
Fabiato A, Fabiato F. Dependence of the contractile activation of
skinned cardiac cells on the sarcomere length. Nature 1975;256:54–
56.
Babu A, Sonnenblick E, Gulati J. Molecular basis for the influence
of muscle length on myocardial performance. Science 1988;240:74–
76.
Tavi P, Han C, Weckstrom M. Mechanisms of stretch-induced
changes in [Ca 21 ]I in rat atrial myocytes: role of increased troponin
C affinity and stretch-activated ion channels. Circ Res
1998;83:1165–1177.
Han C, Tavi P, Weckstrom M. Role of the sarcoplasmic reticulum in
the modulation of rat cardiac action potential by stretch. Acta
Physiol Scand 1999;167:111–117.
Vahl CF, Timek T, Bonz A et al. Length dependence of calciumand force-transients in normal and failing human myocardium. J
Mol Cell Cardiol 1998;30:957–966.
677
[32] Weil J, Eschenhangen T, Hirt S et al. Preserved Frank–Starling
mechanism in human end-stage heart failure. Cardiovasc Res
1998;37:541–548.
[33] Holubarsch C, Ruf T, Goldstein DJ et al. Existence of the Frank–
Starling mechanism in the failing human heart. Investigations on the
organ, tissue, and sarcomere levels. Circulation 1996;94:683–689.
[34] Schwinger RH, Bohm M, Koch A et al. The failing human heart is
unable to use the Frank–Starling mechanism. Circ Res
1994;74:959–969.
[35] Koss KL, Grupp IL, Kranias EG. The relative phospholamban and
SERCA2 ratio: a critical determinant of myocardial contractility.
Basic Res Cardiol 1997;92(Suppl. 1):17–24.
[36] Hajjar RJ, Schmidt U, Kang JX, Matsui T, Rosenzweig A.
Adenoviral gene transfer of phospholamban in isolated rat cardiomyocytes. Rescue effects by concomitant gene transfer of
sarcoplasmic reticulum Ca(21)-ATPase. Circ Res 1997;81:145–
153.
[37] Brittsan A, Kranias EG. Phospholamban and cardiac contractile
function. J Mol Cell Cardiol 2000;32:2131–2139.
[38] Carr AN, Kranias EG. Thyroid hormone regulation of calcium
cycling proteins. Thyroid 2002;12:453–457.
[39] Grupp IL, Subramaniam A, Hewett TE, Robbins J, Grupp G.
Comparison of normal, hypodynamic, and hyperdynamic mouse
hearts using isolated work-performing heart preparations. Am J
Physiol 1993;265:H1401–1410.
[40] Vahl CF, Bonz A, Timek T, Hagl S. Intracellular calcium transient
of working human myocardium of seven patients transplanted for
congestive heart failure. Circ Res 1994;74:952–958.
[41] Pieske B, Maier LS, Bers DM, Hasenfuss G. Ca 21 handling and sR
Ca 21 content in isolated failing and nonfailing human myocardium.
Circ Res 1999;85:38–46.
[42] Munch G, Bolck B, Brixius K et al. SERCA2a activity correlates
with the force–frequency relationship in human myocardium. Am J
Physiol 2000;278:H1924–H1932.
[43] Frank K, Bolck B, Bavendiek U, Schwinger RHG. Frequency
dependent force generation correlates with sarcoplasmic calcium
ATPase activity in human myocardium. Basic Res Cardiol
1998;93:405–411.
[44] Schillinger W, Lehnart SE, Prestle J et al. Influence of SR Ca 21 ATPase and Na 1 –Ca 21 -exchanger on the force–frequency relation.
Basic Res Cardiol 1998;93(Suppl. 1):38–45.
[45] Kadambi VJ, Ball N, Kranias EG, Walsh RA, Hoit BD. Modulation
of force–frequency relation by phospholamban in genetically engineered mice. Am J Physiol 1999;276:H2245–H2250.
[46] Bluhm WF, Kranias EG, Dillmann WH, Meyer M. Phospholamban:
a major determinant of the cardiac force–frequency relationship. Am
J Physiol 2000;278:H249–H255.
[47] Schillinger W, Janssen PM, Emami S et al. Impaired contractile
performance of cultured rabbit ventricular myocytes after adenoviral
gene transfer of Na(1)–Ca(21) exchanger. Circ Res 2000;87:581–
587.