Am J Physiol Heart Circ Physiol 285: H90-H96, 2003.
First published March 13, 2003; doi:10.1152/ajpheart.01018.2002
0363-6135/03 $5.00
Age-related functional effects linked to phosphatase activity in ventricular myocytes
Elizabeth M. Grey,
Chun K. Chan,
Yi Chen, and
Polly A. Hofmann
Department of Physiology, University of Tennessee, Memphis, Tennessee
38163
Submitted 25 November 2002
; accepted in final form 12 March 2003
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ABSTRACT
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Conflicting reports exist regarding the influence of 
-adrenergic
stimulation on the maximum velocity of shortening (Vmax)
in ventricular myocytes. This may be due to an unrecognized effect of
maturation. In the present study, the effects of -adrenergic receptor
stimulation on myocytes from hearts of juvenile nonbred and young adult
retired breeder female rats were compared. Ventricular myocytes from young
adults had a -adrenergic-dependent increase in Vmax
and Ca2+-dependent actomyosin ATPase that was not observed in
myocytes from juveniles. Myocytes from young adults had both an increase in
-myosin heavy chain (MHC) and higher basal serine/threonine phosphatase
activity compared with juvenile rats. Additional studies established moderate
increases in -MHC induced by hypothyroidism do not confer myocardial
-adrenergic responsiveness, whereas inhibition of the higher phosphatase
activity in myocytes from young adults blocks the age-dependent,
-adrenergic-induced increase in cross-bridge cycling rates. We propose
that the higher phosphatase activity of myocytes from young adults compared
with juveniles allows for a greater functional response of the myocardium to
-adrenergic stimulation.
-adrenergic; heart; maturation; velocity of shortening
-ADRENERGIC RECEPTOR STIMULATION of the heart has both
positive inotropic and lusitropic effects. These effects have been primarily
attributed to changes in the duration of Ca2+ transients (for
reviews, see Refs. 1 and
17). Evidence suggests that
the rates of actin-myosin association and dissociation also influence the
rates of myocardial contraction and relaxation. For example,
-adrenergic-induced increases in the relaxation rate may be due, in
part, to an increase in the rate of dissociation of Ca2+ from
troponin C caused by phosphorylation of troponin I
(15).
-Adrenergic-induced increases in the rate of contraction may also be
due, in part, to an increased rate of actin-myosin cycling. However, studies
examining the influence of -adrenergic-protein kinase A (PKA)
stimulation on cardiac cross-bridge cycling rates in myocardial preparations
appear to conflict (3,
7,
8,
18).
De Tombe and ter Keurs (3)
demonstrated that -adrenergic receptor stimulation did not alter the
maximum unloaded sarcomere shortening velocity (Vmax) in
rat intact trabeculae. Consistent with this, Hofmann and Lange
(7) demonstrated that PKA
exposure did not affect Vmax in skinned ventricular
myocytes. However, Strang et al.
(18) found that
-adrenergic receptor activation increased Vmax in
ventricular myocytes from rats. In addition, Hoh et al.
(8) using analysis of the
frequency at which stiffness of the muscle is at a minimum (fmin),
thought to reflect the rate of cross-bridge cycling, found a -adrenergic
receptor-dependent increase in fmin in papillary muscle. The
disparity in findings may have been the result of strain, age, gender, or
breeding history differences. Thus the goals of the present study were to
examine the effect, if any, of age/breeding history on the ability of the
-adrenergic-PKA pathway to influence cross-bridge cycling rates of
ventricular myocytes and to link the observed effects with either
developmental changes in myofilament protein expression or the level of
myofilament protein phosphorylation.
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MATERIALS AND METHODS
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Animals. Hearts from juvenile and young adult Wistar female rats
were used to examine the role of maturation on -adrenergic receptor
activation on cross-bridge cycling rates in isolated ventricular myocytes.
Female juvenile rats were 3–4 mo of age, weighed 245–295 g, and
had not been bred. Juvenile rats in the present study were of similar strain,
age, weight, and breeding history as those used in the study of Hofmann and
Lange (7). Female young adult
rats were 9–10 mo of age, weighed 340–380 g, and were retired
breeders. Retired breeders, rather than nonbred adults, were used to duplicate
conditions of the studies of Strang et al.
(18). Similar conditions to
past studies (7,
18) were used to establish or
refute the existence of a developmental difference on the effect of
-adrenergic stimulation on Vmax and rule out rat
strain or technical variance as the cause for differences in the results of
past studies. Experiments using animals were reviewed and approved by the
University of Tennessee.
Enzymatic isolation of ventricular myocytes. Ventricular myocyte
isolation procedures were identical to Lester et al.
(11). Quiescent, rectangular
myocytes in Ringer solution containing 1.25 mM Ca2+ accounted for
>50% of the total cells.
32P incorporation. Isolated myocytes were exposed to
[32P]orthophosphate for 1 h. Myocytes were then treated for 5 min
with 100 nM isoproterenol (a -adrenergic receptor agonist), untreated,
or exposed for 20 min to 4.5 mM dibutyryl-cAMP (a cell-permeable activator of
PKA). Dibutyryl-cAMP was used at a high concentration to facilitate and speed
entry into the cytosol. After drug treatment, cells were exposed to a relaxing
solution (11) containing 0.3%
Triton X-100 for 6 min. A urea-containing sample buffer was added, and
electrophoretic separation of proteins was carried out
(5,
11).
Myofibrillar Ca2+-dependent ATPase. After enzymatic
isolation, ventricular myocytes were pretreated with 1 µM okadaic acid or
vehicle for 20 min and exposed to 100 nM isoproterenol or vehicle for 5 min.
Myofibrils were then isolated from these cells using the protocol of Murphy
and Solaro (12). Myofibrils
were stored in an ice-cold phosphate buffer solution plus 100 nM calyculin A
to stop all phosphatase activity. Ca2+-dependent actomyosin ATPase
was determined as previously described
(14). In brief, myofibrils
were incubated at 32°C in solutions containing calcium of either high (pCa
4.0) or low (pCa 9.0) concentrations plus 5 mM MgCl2, 3 mM ATP, 2
mM EGTA, 20 mM imidazole (pH 7.0), and enough KCl to bring the final ionic
strength to 60 mM. After a 10-min incubation, the reaction was quenched with
20% trichloroacetic acid, and inorganic phosphate levels were determined
according to the method of Fiske and SubbaRow. Protein concentration was
determined with a Biuret assay. Inorganic phosphate production was found to be
linear with respect to time under conditions of 32°C with a final protein
concentration <2.0 mg/ml (data not shown).
Western blot analysis of -myosin heavy chain.
Ventricles were excised, homogenized, and centrifuged, and the pellet was
immediately washed and placed on ice. Samples were electrophoresed on SDS-PAGE
gels using the methods of Fritz et al.
(5) or Talmadge and Roy
(19). Both methods of SDS-PAGE
utilized a 5% acrylamide stacking gel. The acrylamide resolving gel was either
a 12% gel at a 200:1 acrylamide-to-bis ratio, with 0.75 M Tris at pH 9.3, 0.1%
SDS, and 10% glycerol (5), or
an 8% gel at a 50:1 acrylamide-to-bis ratio, with 0.2 M Tris at pH 8.8, 0.4%
SDS, and 5% glycerol (19). The
two methods were used to examine the effect of optimizing myosin heavy chain
(MHC) separation. Gel proteins were transferred to polyvinylidene difluoride
(PVDF) membranes. Coomassie staining of SDS gels and blots indicated the
protein load and extent of transfer of MHC to PVDF membrane were similar in
myocardial samples from juvenile and young rats.
Immunoblotting was performed as described in the protocol included with
NEN/DuPont chemiluminescent reagents (NEL-102, NEN/DuPont). PVDF membranes
were incubated with either a monoclonal anti--MHC antibody isolated
against skeletal muscle slow MHC (1:100 dilution, catalog number MAB1628,
Chemicon International; Temecula, CA), a monoclonal anti--MHC antibody
isolated from an immunogen of human ventricular myosin and recognizing a light
meromyosin fragment (1:50 dilution, catalog number MAB1552, Chemicon
International), or a monoclonal anti-
-MHC antibody isolated from an
immunogen of purified human atrial myosin (1:10 dilution, catalog number
BS-1170-S, Alexis).
Induction of hypothyroidism. Juvenile rats, 3 mo of age, were
given 0.8 mg/ml propylthiouracil (PTU) in their drinking water for 2 or 4
days.
Velocity of unloaded shortening. Isolated myocytes were treated
for 5 min with 100 nM isoproterenol, untreated, or treated for 20 min with 4.5
mM dibutyryl-cAMP. Myocytes were then skinned with a relaxing solution
containing 0.3% Triton X-100, washed, and placed on ice.
Vmax was determined using equipment and procedures
identical to Hofmann and Lange
(7). In brief, a cell was glued
at its ends to micropipettes projecting from a piezo electric translator and
force transducer. Sarcomere length was set to 
2.2 µm using a
microscope micrometer. Cells are activated in a maximally activating
Ca2+-containing saline solution of pCa 4.5
(7). Once steady-state tension
was achieved, cells were slackened by a given amount, causing tension to fall
to zero. Tension redevelopment was observed when the activated cell had take
up the imposed slack. Multiple slackening steps were induced in one cell and
plotted as a change in length (slack length) versus duration of unloaded
shortening (time until onset of tension redevelopment). The slope of this
relationship, in muscle lengths per second, is a measure of
Vmax. Data from cells that met the following criteria were
included in the final analysis: striations were clearly visible in the
photomicrographs taken in pCa 9.0 and pCa 4.5 solution, sarcomere length
between these pCa solutions differed by <0.25 µm, final maximum
contraction value was 70% or greater of the initial maximum contraction value,
and the goodness of fit to the slack versus duration plot was ≥0.80.
Statistics. All values are reported as means ± SE, and
P < 0.05 was chosen to indicate statistical significance. All data
were analyzed by ANOVA and Fisher's least-significant difference post hoc
test.
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RESULTS
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Age on -adrenergic-dependent functional effects.
Vmax was determined in myocytes treated and subsequently
skinned from juvenile and young adult female rats. Pretreatment of intact
myocytes with either isoproterenol or dibutyryl-cAMP did not alter
Vmax in ventricular myocytes from juvenile female rats
(Fig. 1), but increased
Vmax in myocytes from young adult female rats.
Vmax was not significantly different between unstimulated
(control) myocytes from juvenile and young adults.
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Fig. 1. Cumulative maximum velocity of unloaded shortening
(Vmax) comparing control (Con), 100 nM isoproterenol
(Iso)-treated, and 4.5 mM dibutyryl-cAMP-treated ventricular myocytes.
Myocytes were isolated from hearts of rats that were either juvenile (3 mo
old) or young adults (9–10 mo old). Values are expressed as means
± SE. *P < 0.05 compared with same-age control
myocytes.
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Isometric tension as a function of Ca2+ concentration was also
determined in myocytes treated and subsequently skinned from juvenile and
young adult female rats. Maximum isometric tension did not change in any of
the preparations or treatment groups (Table
1). Ca2+ sensitivity of tension as determined by the
negative log of the Ca2+ concentration at which 50% tension is
observed (pCa50) increased after PKA activation to a similar extent
in myocytes from juvenile and young adult female rats
(Table 1 and
Fig. 2). No significant
differences in the Hill coefficients (steepness) of the tension-pCa
relationship occurred between any groups
(Table 1).
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Fig. 2. Cumulative tension-pCa relationships comparing Con, 100 nM Iso-treated, and
4.5 mM dibutyryl-cAMP-treated ventricular myocytes. Myocytes were isolated
from hearts of rats that were either juveniles (A) or young adults
(B). Additional information and statistics can be found in
Table 1 and in the text. Values
are expressed as means ± SE.
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Role of phosphorylation on age effects. Dibutyryl-cAMP increased
phosphorylation of C protein and troponin I in ventricular myocytes from
juvenile and young adult female rats (Fig.
3). Isoproterenol stimulation also increased C protein and
troponin I phosphorylation in myocytes from young adults with a trend to
increased phosphorylation observed in myocardium from juvenile female rats
compared with controls (Fig.
3). Myocytes from young adults had a significantly lower level of
phosphorylation of troponin I and C-protein under all conditions compared with
myocytes from juvenile rats.
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Fig. 3. Cumulative analysis of phosphate incorporation into troponin I (A)
and C protein (B) normalized to the Coomassie stain density.
Ventricular myocytes were isolated from hearts of rats that were either
juvenile (n = 7) or young adults (n = 6). 32P
incorporation of myocytes was brought about by exposure to 100 nM Iso and 4.5
mM dibutyryl-cAMP. Values are expressed as means ± SE.
*P < 0.05 compared with same-age control myocytes;
 P < 0.05 compared with different age but same treatment
group.
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In a separate series of experiments, we confirmed that ventricular myocytes
from young adults had a significantly lower level of basal/unstimulated
phosphate incorporation into troponin I and C protein compared with myocytes
from juvenile rats (Fig. 4). Basal phosphorylation of troponin I was unaffected by PKA inhibition with H89
in myocytes from both juveniles and young adults, whereas phosphatase
inhibition using 1 µM okadaic acid increased basal phosphorylation of
troponin I in myocytes from young adults but not juveniles
(Fig. 4).
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Fig. 4. Cumulative analysis of basal phosphate incorporation into troponin I
normalized to the Coomassie stain density. Ventricular myocytes were untreated
(Con), exposed to 10 µM H89, or exposed to 1 µM okadaic acid (Oka) for
20 min. H89 is a protein kinase A inhibitor, whereas okadaic acid inhibits
protein phosphatase 1 and 2a. Myocytes were isolated from hearts of rats that
were either juveniles (n = 3) or young adults (n = 3).
Values are expressed as means ± SE. *P < 0.05
compared with same-age control myocytes; P < 0.05 compared
with same treatment.
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To establish the contribution of phosphatase activity to -adrenergic
reactivity, Ca2+-dependent actomyosin ATPase was determined in
myocytes that were pretreated with 1 µM okadaic acid or vehicle and then
stimulated with isoproterenol or vehicle, and myofibrils isolated. In the
absence of phosphatase inhibition, isoproterenol did not alter
Ca2+-dependent actomyosin ATPase in cardiac myofibrils from
juvenile female rats (Fig.
5A) but increased Ca2+-dependent actomyosin
ATPase in cardiac myofibrils from young adult female rats
(Fig. 5B). In the
presence of phosphatase inhibition, isoproterenol did not increase
Ca2+-dependent actomyosin ATPase in cardiac myofibrils from either
juvenile or young adult female rats compared with controls. Myofibrils
isolated from young adult rat hearts that were pretreated with okadaic acid
had a significantly higher Ca2+-dependent actomyosin ATPase than
those not treated with okadaic acid (Fig.
5B).
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Fig. 5. Cumulative Ca2+-dependent actomyosin ATPase comparing Con and
100 nM Iso-treated ventricular myocytes in the presence (+) or absence (-) of
1 µM Oka. Myocytes were isolated from hearts of rats that were either
juvenile (A) or young adults (B), cells were treated as
noted, and myofibrils were isolated. Values are normalized to Con myofibrils
not treated with Oka and expressed as means ± SE; n = 6
isolations for all groups. *P < 0.05 compared with Con
myocytes of the same treatment group; P < 0.05 compared with Con
myocytes with no Oka treatment.
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Role of -MHC on age effects. -MHC expression
was higher in ventricular myocardium from young adult compared with juvenile
female rats. Figure 6 presents
typical Western blots using MHC antibodies against ventricular myocardium from
juvenile (J1–J3) and young adult rats (A1–A3). Membranes were
incubated with either a monoclonal anti--MHC
(Fig. 6A,
top) or a monoclonal anti--MHC antibody
(Fig. 6, A,
bottom, and B). Myocardial samples from five young adult and
four juvenile rats gave similar results as those shown in
Fig. 6.
Myocardial samples were also electrophoresed using a protocol that
optimizes MHC separation (Fig.
6B) (13).
Two -MHC-reactive bands were observed in myocardium from young adults.
This was consistently observed using either a monoclonal antibody directed to
slow skeletal muscle -MHC (Fig.
6B) or a monoclonal antibody directed to the light
meromyosin portion of -MHC. The lower-molecular-mass band in
Fig. 6B did not
increase in density in samples intentionally processed to increase proteolysis
by leaving the homogenized heart at room temperature for 3 h (data not
shown).
To determine the contribution of -MHC to -adrenergic
reactivity, short-term hypothyroidism was induced. Juvenile rats that had
access to drinking water with 0.8% PTU for 4 days had an increase in the
-MHC-immunoreactive band similar
(Fig. 7A) to that seen
in young adult rats (Fig.
6A). Ventricular myocytes from these rats were stimulated
with isoproterenol or vehicle, and myofibrils were isolated. Isoproterenol did
not alter the Ca2+-dependent actomyosin ATPase of myofibrils from
either euthyroid (control) or hypothyroid (PTU) juvenile rats
(Fig. 7B).
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Fig. 7. A: -MHC content in cardiac homogenates from juvenile rats
treated with propylthiouracil (PTU) for 2 or 4 days. B: cumulative
Ca2+-dependent actomyosin ATPase comparing Con and 100 nM
Iso-treated ventricular myocytes of Con and PTU-treated juvenile rats.
Myofibrillar ATPase data were obtained from myocytes isolated from hearts of
juvenile rats that had access to plain drinking water (Con rats) or water with
0.8 mg/ml PTU for 4 days (PTU rats). Values are normalized to Con myofibrils
from Con rats and expressed as means ± SE; n = 3 isolations
for all groups. No statistically significant differences were found.
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DISCUSSION
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The present study demonstrates the existence of an age/breeding
history-dependent, -adrenergic-cAMP-induced increase in
Vmax in cardiac myocytes. This observation is novel and
may explain apparent contradictions in the literature regarding the effect of
the -adrenergic pathway on cardiac Vmax and
cross-bridge interaction rates
(3,
7,
8,
18). In the present study
(summarized in Fig. 8), a
higher basal phosphatase activity in hearts from young adults compared with
juveniles was demonstrated by a decrease in phosphorylation of endogenous
phosphoproteins troponin I and C protein. Elimination of the higher
phosphatase levels in myocytes from young adults blocked the ability of
-adrenergic stimulation to increase cross-bridge interaction rates.
Age-dependent increases in -MHC did not appear to confer
-adrenergic responsiveness. Thus we propose developmental changes in
serine/threonine phosphatase activity can influence the functional reactivity
of myocardium to stimuli.
Past studies have demonstrated that stimulation of the -adrenergic
pathway increases (8,
18) or causes no change
(3,
7) in the cross-bridge cycling
rate. We hypothesized that the apparent conflict in the data arose out of age
or breeding history differences. For example, Vmax data
from cardiac myocytes of 3-mo-old females that were not bred
(7) appears to conflict with
data using 8- to 9-mo-old retired breeder females
(18). The present study
confirms the hypothesis that age/breeding history has an effect on
-adrenergic-induced changes in cardiac myocyte Vmax
and actomyosin ATPase.
Maximum tension was not affected by activation of the -adrenergic-PKA
pathway in myocytes from either juvenile or young adults. This finding is
consistent with previous studies using skinned myocytes
(7,
18). A decrease in the
Ca2+ sensitivity of tension in all myocytes was seen on
-adrenergic receptor stimulation and dibutyryl-cAMP exposure. The
magnitude of the -adrenergic-dependent decrease in pCa50 was
not statistically different in myocytes from juveniles and young adults.
-Adrenergic-induced decreases in Ca2+ sensitivity of tension
have previously been shown to be related to troponin I phosphorylation
(6).
Maturation of rats from 3 to 9 mo of age caused a 7–15% decrease in
-MHC with similar increase in -MHC
(2,
4). Our findings are
qualitatively consistent with these observations. In addition, we subjected
juvenile rats to short-term hypothyroidism to cause a modest increase in
-MHC independent of the aging process. We observed that
Ca2+-dependent actomyosin ATPase activity was unchanged in
isoproterenol-stimulated cardiac myocytes from hypothyroid and euthyroid
juvenile rats. Thus increases in -MHC do not appear to account for the
observed -adrenergic-dependent increase in cross-bridge cycling rates of
cardiac myocytes from young adult rats.
Norepinephrine-induced phosphorylation of cardiac troponin I is reduced in
hearts of 24- versus 6-mo-old rats
(10). However, this study and
others (9,
16,
20) were designed to examine
the role of senescence (>24 mo of age in rats) rather than
maturation/development. We are unaware of any past study that compares the
levels of basal or -adrenergic-induced phosphorylations of cardiac
myofilament proteins in juvenile and young adult rats. In the present study, a
decrease in the extent of phosphorylation of troponin I and C protein occurred
in myocytes from young adults under basal and -adrenergic-stimulated
conditions compared with juveniles. An age-dependent decrease in the
-adrenergic receptor-adenylate cyclase pathway was not responsible for
this observation since both isoproterenol stimulation, a -adrenergic
receptor agonist, and direct stimulation of PKA with dibutyryl-cAMP lead to
lower stimulated levels of myofilament phosphate incorporation in young adults
compared with juveniles.
The decrease in basal phosphorylation of myocytes from young adults could
be due to decreased kinase activity or increased phosphatase activity under
resting conditions. Our results using phosphatase and PKA inhibitors
demonstrate that only phosphatase inhibitors have a significant effect on
basal phosphorylations of troponin I and C protein in myocytes from young
adults. Basal myofilament protein phosphorylation in juveniles was not altered
by either phosphatase or PKA inhibitors. These observations indicate that
young adults have a higher level of myocardial phosphatase activity than
juveniles rats. Past studies have demonstrated that activation of estrogen
receptors on cardiac myocytes increase phosphatase expression and may
contribute to gender-based differences in cardiac disease
(13). This raises the
possibility that in our studies the increased phosphatase activity in
myocardium of young adult, retired breeder, female rats was due to hormones
associated with past pregnancies or menstrual cycling rather than age in and
of itself.
Ca2+-dependent actomyosin ATPase activity increased with
isoproterenol stimulation of cardiac myocytes from young adult rats.
-Adrenergic stimulation of myocytes from juvenile rats did not affect
the Ca2+-dependent actomyosin ATPase activity. These findings are
consistent with our measurements of Vmax. In addition,
myocytes from young adults that were pretreated with a phosphatase inhibitor
had an increase in basal actomyosin ATPase and an inhibition of
-adrenergic receptor-dependent increase in Ca2+-dependent
actomyosin ATPase activity. Cardiac myocytes and isolated myofibrils from
juvenile rats demonstrated no change in actomyosin ATPase with phosphatase
inhibition. These findings suggest that age-and/or breeding history-dependent
increases in phosphatase activity enhance the functional response of the
myocardium to -adrenergic stimulation as animals mature from juveniles
to young adults.
The mechanism by which increased phosphatase activity allows for
-adrenergic responsiveness in myocytes is unclear. One possibility is
that there is a myocardial protein that is phosphorylated, by a kinase other
than PKA, under resting conditions, and that this phosphorylation inhibits
PKA-dependent effects. Under conditions of high basal phosphatase activity,
this inhibition is removed and -adrenergic-PKA activation leads to an
increase in the rate of actomyosin association. More simply stated, a higher
phosphatase activity changes the "gain" on the system to unmask a
-adrenergic effect. Our data are consistent with such a mechanism, but
do not identify the hypothesized inhibitory protein. What can be definitively
stated from our studies is that Ser/Thr phosphatases are not static
"housekeeping" enzymes but modulate how second messengers
influence cardiac function.
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ACKNOWLEDGMENTS
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This study was supported by National Heart, Lung, and Blood Institute Grant
HL-48839 and an American Heart Association Established Investigatorship (to P.
A. Hofmann).
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FOOTNOTES
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Address for reprint requests and other correspondence: P. A. Hofmann, Dept. of
Physiology, Univ. of Tennessee, 894 Union Ave., Memphis, TN 38163 (E-mail:
phofmann{at}physio1.utmem.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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Copyright © 2003 by the American Physiological Society.
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