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Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Normal aging of
the rodent heart results in prominent prolongation of the twitch. We
tested the hypothesis that increased expression of 
-myosin heavy
chain (MHC), as occurs in the normal aging process in the rodent heart,
contributes to the prolongation of the twitch by depressing the
kinetics of cross-bridge interaction. Using 3-, 9-, 21-, and 33-mo-old
male Fischer 344 × Brown Norway F1
hybrid rats, we examined both the rate of tension development (kCa) and
unloaded shortening velocity in chemically skinned myocardium. Although
kCa in all four
age groups was dependent on the level of
Ca2+ activation, both submaximal
and maximal kCa
were significantly slower in 9-, 21-, and 33-mo-old rats relative to
3-mo-old rats. Furthermore, unloaded shortening velocity was
significantly reduced in 9-, 21-, and 33-mo-old rats compared with
3-mo-old rats. Collectively, these data strongly suggest that the
aging-related increase in -MHC expression results in a progressive
slowing of cross-bridge interaction kinetics in skinned myocardium,
which most likely contributes to the overall aging-dependent reduction
in myocardial functional capacity.
myosin heavy chain; caged calcium; cross-bridge kinetics; Fischer 344 × Brown Norway rats
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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THE TRANSITION from adulthood to senescence is
characterized by a progressive decline in cardiac function,
particularly in the context of acute increases in myocardial functional
load, as in exercise. The depression of cardiac contraction in
senescence is evident as a prominent slowing of twitch and
Ca2+ handling kinetics.
Contraction duration, time to peak tension, and half- time for
relaxation are significantly prolonged in senescent myocardium relative
to values observed in juvenile animals (10, 17, 36) and are mediated at
least in part by an aging-dependent slowing of
Ca2+ uptake by the sarcoplasmic
reticulum (SR) (11). The intracellular Ca2+ transient has been shown to
be significantly prolonged with advancing age (29), an effect that may
be related to a depressed SR Ca2+
pump rate (11). In addition, the capacity for -adrenergic modulation
of SR function is progressively attenuated with increasing age. Several
factors may account for this age-related effect, including a decrease
in the level of intracellular cAMP (17, 36) and a diminished
transsarcolemmal Ca2+ current
after -adrenergic stimulation (49).
Prolonged contraction time may also result from modulation of the
kinetics of cross-bridge interaction, due to possible changes in myosin
heavy chain (MHC) profile, among other factors. Support for a role of
MHC stems from the work of Metzger and Moss (23), who demonstrated that
the maximal rate of tension redevelopment was nearly eightfold greater
in fast-twitch relative to slow-twitch skeletal muscle fibers. The
transition to a strongly bound force-generating state at high
Ca2+ concentration was well
correlated with the fiber type-specific expression of skeletal muscle
MHC isoforms. Variations in maximal shortening velocity, an index of
cross-bridge detachment rate, are also thought to be related to the
expression of ventricular MHC isoforms (30). Furthermore, in vitro
motility studies have shown that the addition of only moderate amounts
of -MHC to 
-MHC preparations can dramatically reduce sliding
velocity to values similar to those seen in preparations containing
100% -MHC (14). The normal aging process in the rodent heart is
associated with a significant age-induced alteration in ventricular MHC
expression from ~80% -MHC/20% -MHC at 3 mo to
~30% -MHC/70% -MHC by 24-26 mo (4, 22).
To date, no studies have investigated the effects of variable
ventricular MHC isoform expression on the rates of activation and
relaxation of force in myocardium, especially in the context of normal
aging. The age-dependent increase in -MHC expression and consequent
effects on myocardial contractile function in the rodent heart may even
be relevant in the context of human aging in light of recent evidence
demonstrating significant amounts of -MHC mRNA transcripts in the
normal human left ventricle and a marked decrease in -MHC
transcripts in chronic heart failure (25). Therefore, this study was
designed to investigate possible effects of the age-induced increase in
-MHC expression on kinetics of cross-bridge interaction in
ventricular myocardium.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Animal Care
Virgin male Fischer 344 × Brown Norway (F344×BN) F1 hybrid rats were obtained from the National Institute on Aging animal colony maintained by Harlan Sprague-Dawley (Indianapolis, IN). F344×BN rats were used in this study because the F1 hybrid does not exhibit many of the age-related pathologies commonly seen in the aged Fischer 344 and Wistar rats (20). Animals were selected to represent a continuum of ages, including juvenile (i.e., 3 mo), young adult (i.e., 9 mo), middle-aged (i.e., 21 mo), and senescent (i.e., 33 mo) rats. The rats were housed in individual plastic cages in a positive-air flow room that was maintained at 21-22°C. The rats were fed NIH 31 rat food and water ad libitum and maintained on a 12-h:12-h light-dark cycle. All animals were allowed to acclimatize for a minimum of 10 days before experimentation. Every animal was inspected daily for external signs of disease throughout the course of the study. No rats were excluded from use in this study. All animal usage was conducted under the strict guidelines established by the University of Wisconsin Animal Care Committee.Experimental Solutions
The isolated working heart preparation was perfused with a Krebs-Henseleit buffer containing the following (in mM): 118 NaCl, 25 NaHCO3, 5 glucose, 4.8 KCl, 2.6 CaCl2, 1.2 MgSO4, and 1.2 KH2PO4 (28). Compositions of relaxing and activating solutions used in the mechanical experiments were as follows (in mM): relaxing solution, 100 KCl, 20 imidazole, 4 ATP, 2 EGTA, and 1 free Mg2+, pH 7.0 at 22°C; activating solutions, 79 KCl, 20 imidazole, 14.5 creatine phosphate, 7 EGTA, 5.4 MgCl2, 4.7 ATP, and free Ca2+ ranging from 1 nM (i.e., pCa 9.0) to 32 µM (i.e., pCa 4.5). The compositions of solutions used in flash photolysis experiments are listed in Table 1. A computer program (8) was used to calculate the final concentrations of each metal, ligand, and metal-ligand complex based on stability constants described by Godt and Lindley (13).
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Tissue Preparation
Animals used in mechanical experiments were anesthetized by placing them in a glass bell jar containing room air and 4% methoxyflurane. Deep anesthesia was confirmed by the loss of the pedal reflex and muscular tension of the limbs. All animals were killed by creating a pneumothorax after the establishment of deep anesthesia by inhalation of methoxyflurane, and the heart of each rat was rapidly excised and trimmed free of atria and great vessels. The left ventricle (LV) was blotted dry and weighed. Approximately 300-400 mg of LV tissue were placed in a beaker of ice-cold relaxing solution and cut into 5- to 10-mm pieces. The remaining LV sample (~300-700 mg) was placed in a cryotube and stored at
80°C until analyzed for
MHC distribution by SDS-PAGE. The minced LV sample was homogenized for
4 s in a Polytron homogenizer to yield multicellular bundles
(dimensions: 600-900 µm × 100-180 µm) or for 8 s to
yield single myocytes (dimensions: ~100 µm × 20 µm). The
cellular homogenate was centrifuged at 120 g for 1 min and resuspended twice in
fresh relaxing solution. After the final spin, the pelleted myocardial preparations were rapidly and completely chemically skinned by resuspending the pellet for either 6 min (single myocytes) or 30 min
(multicellular bundles) in fresh relaxing solution containing 0.3%
Triton X-100. The skinned myocardial preparations were washed twice
with fresh relaxing solution and stored on ice until used.
Experimental Procedures
Isolated working heart preparation.
Rats were anesthetized with ketamine hydrochloride (90 mg/kg ip) to
induce deep anesthesia, the thoracic cavity of each rat was opened by
midline sternotomy, and the heart was rapidly excised. The proximal
aorta was cannulated, and the heart was perfused in a retrograde manner
with Krebs-Henseleit buffer (37°C, preequilibrated with 95%
O2-5%
CO2) at a constant perfusion
pressure of 100 mmHg (28). A small incision was made in the left
atrium, and a small, thin latex balloon tied to the end of a section of
PE-190 tubing was advanced across the mitral valve and placed in the LV
chamber. The PE-190 tubing was secured to the left atrial tissue. Heart rate was paced at 390 beats/min throughout the time course of the
experiment with electrodes sutured to the right atrium. A heart rate of
390 beats/min is well within the normal in vivo levels of a resting rat
(9). LV volume was adjusted by infusion of degassed saline into the
latex balloon (offset by the measured volume of the empty balloon). LV
pressure was measured using an ultraminiature pressure transducer
(3-Fr; Millar Instruments, Houston, TX), which was tied so that the tip
was within the latex balloon. Indexes of myocardial function including
peak LV systolic pressure, LV end-diastolic pressure, and the rates
of LV pressure development (LV
+dP/dt) and relaxation (LV
dP/dt) were recorded continuously. After a 10- to 15-min equilibration period, LV pressures were measured over a range of ventricular volumes corresponding to LV
end-diastolic pressures of 0-20 mmHg. Calculated measures of LV
systolic function include the slope of the isovolumic end-systolic pressure-volume relation and the slope of the relationship between LV
+dP/dt and LV volume (48). Coronary
resistance at each LV volume was calculated by measuring coronary flow
at a constant coronary perfusion pressure of 100 mmHg.
Tension-pCa relationship. An experimental apparatus similar to one described previously (40) was used to attach single skinned ventricular myocytes and to record steady-state submaximal (P) and maximal Ca2+ activated tensions (P0), the Ca2+ sensitivity of tension (pCa50), and unloaded shortening velocity (V0). Myocytes were attached with silicone adhesive to steel pins (10 µm OD) that were fixed with paraffin wax to a piezoelectric translator (Physik Instrument, Waldbronn, Germany) and a force transducer (model 403, Cambridge Technology, Cambridge, MA). P0 and pCa50 were determined at a sarcomere length of ~2.25 µm. Apparent cooperativity of tension development was estimated from the steepness of the tension-pCa relationship for Ca2+-activated tensions <0.5 P0 (i.e., nH, Hill coefficient), which was quantified using Hill plot transformations of the tension-pCa data (40). We focused on this region of the curve because the tension-pCa relationship is biphasic, and most of the cooperative activation of the thin filament is evident at tensions <0.5 P0 (24).
Tension-pCa relationships were obtained by first maximally activating the myocyte in a solution of pCa 4.5 and then transferring the myocyte to a series of submaximal pCa solutions between pCa 6.0 and 5.0. At each pCa, steady-state tension was measured by rapidly slackening the myocyte by 20% of its total length. Ca2+-activated tension at a given pCa was calculated as the difference between the total steady-state tension and the resting tension obtained by slackening the myocyte in a solution of pCa 9.0. To determine any decline in tension-generating capability, myocytes were maximally activated in a solution of pCa 4.5 at the end of each protocol, and the reference P0 value for successive submaximal activations was interpolated between the initial and final measurements of maximal tension. Ca2+-activated tensions (P) obtained in solutions of submaximal pCa were expressed as a fraction of P0, i.e., P/P0. Tension-pCa data were fit using the following equation
![P/P<SUB>0</SUB> = [Ca<SUP>2+</SUP>]<SUP><IT>n<SUB>H</SUB></IT></SUP>/(<IT>k</IT><SUP><IT>n<SUB>H</SUB></IT></SUP> + [Ca<SUP>2+</SUP>]<SUP><IT>n<SUB>H</SUB></IT></SUP>)](/content/vol276/issue5/fulltext/H1511/img001.gif)
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Unloaded shortening velocity. V0 was measured during maximal activation of each myocyte by the slack test method (40). Once steady-state tension was reached, the myocyte was slackened by 16-20% of initial length, starting from a sarcomere length of ~2.25 µm. The time between imposition of a slack step and the redevelopment of force was measured by fitting a horizontal line by eye through the tension baseline and determining its intersection with a straight line drawn through the initial portion of tension redevelopment. The maximum amount of slack imposed was such that the myocyte did not actively shorten below a sarcomere length of 1.80 µm, at which point distortion due to mechanical restoring forces is likely to occur (40). Length change (as percent initial length) was plotted as a function of duration of unloaded shortening (in ms). V0, in muscle lengths per second, was determined from the slope of a line fitted to the data by linear regression analysis. Data from a given myocyte were discarded if the regression coefficient was <0.95.
Rate of tension development after flash photolysis of DM-Nitrophen.
Multicellular skinned preparations were mounted in an experimental
apparatus described previously for skeletal muscle fibers (33). One end
of the preparation was attached to the arm of a torque motor (model
350, Cambridge Technology), and the other end was attached to a force
transducer (model 403, Cambridge Technology). All experiments were
performed using the solutions listed in Table 1 and with sarcomere
length set at ~2.30 µm in relaxing solution. The rate of tension
development was determined after flash photolysis of DM-Nitrophen
(Calbiochem, La Jolla, CA). When exposed to a flash of ultraviolet (UV)
light (
~360 nm), DM-Nitrophen rapidly (<2 ms) releases
Ca2+ due to a decrease in
Ca2+ binding affinity from 5 nM to
3 mM (18). At the beginning of each experiment,
P0 was determined by first bathing
the myocardial preparation in preactivating solution and then in
activating solution of pCa 4.5. Once steady-state force was reached,
the preparation was slackened to achieve a force baseline and then
returned to relaxing solution. The protocol used to determine the rate
of tension development consisted of four steps. The preparation was transferred from relaxing solution to the following sequence of solutions: 1) preactivating solution
for 4 min with two solution changes,
2) loading solution containing 0.4 mM CaCl2 and 1 mM DM-Nitrophen for
5 min, 3) silicone oil (Dow Corning
200 fluid, viscosity = 10 cs) in a ~80-ml quartz trough for recording
changes in force after flash photolysis, and
4) relaxing solution. While the
preparation was in silicone oil, different levels of activation were
achieved by photolyzing DM-Nitrophen with either a low-intensity or
high-intensity UV flash ( ~360 nm; Optoelektronic, Hamburg, Germany). Before the flash, the preparations generated negligible active force, since the calculated free
Ca2+ concentration (pCa 7.0) was
well below the threshold required to activate the myofilaments (Fig.
3). After flash photolysis of DM-Nitrophen, the rate of tension
development
(kCa) was
determined by fitting the experimental data with a single exponential
equation of the form
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Electrophoretic analysis of LV contractile proteins.
The age-related shift in expression of myofibrillar protein isoforms
was determined using 12% SDS-PAGE and ultrasensitive silver staining
after solubilization of single LV bundles in SDS sample buffer (12).
The proportions of ventricular MHC isoforms were determined by
densitometric analysis of silver-stained gels using a GS-670 imaging
densitometer and Molecular Analyst software (Bio-Rad Laboratories,
Hercules, CA). The proportions of -MHC and -MHC were estimated by
expressing the area under the peak for each isoform as a fraction of
the total areas for the two isoforms.
Statistics
All data are expressed as means ± SE. A one-way ANOVA/Bonferroni method was used to determine statistical significance across the four age groups, with significance set at P < 0.05 (46).| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Morphological and Myocardial Functional Adaptations With Aging
Body weight and myocardial mass.
Body weight and myocardial morphological data for each age group (i.e.,
3, 9, 21, and 33 mo) are summarized in Table
2. The age groups used in the present study
represent the spectrum of aging, spanning juvenile (i.e., 3 mo) and
senescent (i.e., 33 mo) stages. Body weight progressively increased as
a consequence of aging, with significant differences noted for the 21- and 33-mo-old rats relative to 3-mo-old rats
(P < 0.05). Total ventricular weight and LV weight were significantly increased in the 21- and 33-mo-old rats compared with 3-mo-old rats (P < 0.05). However, aging did not induce any significant myocardial
hypertrophy, since the ratios of ventricular weight to body weight and
LV to body weight were not significantly different in the 9-, 21-, and
33-mo-old rats compared with 3-mo-old rats.
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LV contractile protein expression.
We observed a dramatic age-dependent increase in the expression of
-MHC and a commensurate downregulation of -MHC expression in the
9-, 21-, and 33-mo-old rats relative to 3-mo-old rats (Table 2). Figure
1A
depicts the age-dependent shift in LV MHC isoform profile. In 3-mo-old
rats, the LV expression of MHC was ~80% -MHC and 20% -MHC. By
33 mo, the level of -MHC expression had significantly increased to
comprise nearly 72% of the total LV MHC, whereas -MHC expression
had decreased to only 28% of total MHC isoform content
(P < 0.05; Table 2). We
did not observe any aging-dependent alterations in the expression of
either the ventricular light chains (i.e.,
MLC1v and
MLC2v) or thin
filament-associated regulatory proteins (Fig.
1B), in agreement with previously
published results (4, 38).
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Ventricular function.
To document an age-related decline in myocardial function capacity, LV
systolic function was obtained from 3- and 33-mo-old rats using an
isolated working heart preparation. Assessment of in vivo cardiac
function is potentially problematic, since LV contractility is
influenced by both preload (i.e., LV end-diastolic volume) and
afterload (i.e., systemic blood pressure). With the use of an isolated
working heart preparation, indexes of LV contractility can be obtained
during isovolumic contractions elicited for a given inotropic state.
Under isovolumic conditions and a paced heart rate of 390 beats/min, LV
functional capacity of the 33-mo-old group was markedly depressed
relative to that seen in the 3-mo-old group at each stage of the
experimental protocol. Peak LV pressure (Fig.
2A) and
LV +dP/dt and LV
dP/dt (Fig.
2B) were substantially reduced in
the 33-mo-old group. The depression of contraction observed in the
33-mo-old rats in the present study is consistent with depressed
myocardial function in senescent rats reported previously (2).
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Activation Dependence of Contraction
Steady-state mechanical measurements.
Table 3 summarizes steady-state mechanical
measurements in single skinned myocytes obtained from 3-, 9-, 21-, and
33-mo-old rats. We observed no significant age-related alterations in
1) maximal
Ca2+-activated tension
(P0),
2) the calcium sensitivity of
tension (pCa50), or
3) the Hill coefficient
(nH) for
Ca2+-activated tensions less than
0.5P0. Figure
3 presents cumulative tension-pCa
relationships for skinned single myocytes as a function of age and
shows that there was no change in the
Ca2+ sensitivity of tension (Table
3, steady-state mechanical
measurements). The lack of age-dependent changes in
the Ca2+ sensitivity of tension is
consistent with results reported previously by Bhatnagar et al. (6) and
provides corroborating evidence that aging did not alter the relative
expression of thin filament-associated regulatory proteins, since
variations in the expression of troponin T (16, 27) have previously
been shown to significantly alter the
Ca2+ sensitivity of tension. We
also found no significant aging-related alterations in the steepness of
the tension-pCa relationship (the Hill coefficient,
nH) at least
within the variability of the data (Table 3), suggesting that MHC
expression has no effect on the apparent cooperativity of tension
development. However, as a consequence of the age-induced increase in
-MHC expression,
V0 progressively decreased in an age-related manner (Table 3). Upregulation of -MHC
expression and concomitant downregulation of -MHC expression have
previously been shown to dramatically reduce
V0 in skinned multicellular preparations from adult myocardium (30, 45).
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Rates of tension development assessed by flash photolysis of
DM-Nitrophen.
The rates of tension development for a given level of activation were
assessed in myocardium from 3-, 9-, 21-, and 33-mo-old rats, and mean
data are presented in Table 3. A representative skinned ventricular
preparation from a 33-mo-old rat was preequilibrated for 5 min in
loading solution containing 0.4 mM
CaCl2 and 1 mM DM-Nitrophen and
then exposed to flashes of low-intensity (Fig. 4, curve
a) and high-intensity (Fig. 4, curve
b) UV light. Before either flash, the preparation
generated negligible active force, since the calculated free
Ca2+ concentration (pCa 7.0) was
well below the threshold for activation of the myofilaments. After
photolysis with a low-intensity UV flash, tension increased to a steady
level of 0.66 P0 with an apparent
rate constant
(kCa) of 3.61 s
1. When the same
preparation was exposed to a high-intensity UV flash, force increased
to a steady level of 0.84 P0 with
a kCa of 5.33 s1. Although force
dependence of the rate of tension development was observed across the
spectrum of ages examined, the rate at which tension developed after
the photorelease of caged Ca2+ was
markedly depressed as a function of increasing age. Because aging did
not influence either the relative distribution of the ventricular
myosin light chain (MLC) isoforms or thin filament-associated regulatory proteins, the results of the present study suggest that the
rates of submaximal and maximal tension development in rat ventricular
myocardium are depressed in accordance with the age-induced increase in
-MHC expression.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The present study was undertaken to determine the impact of
aging-dependent increases in ventricular -MHC expression on kinetics of cross-bridge interaction in skinned myocardial preparations. The
rate of tension development was measured in multicellular ventricular
preparations after flash photolysis of the photolabile Ca2+ chelator DM-Nitrophen, while
V0, an index of
cross-bridge detachment rate, was measured in skinned single myocytes.
The results demonstrate that the age-related increase in -MHC
expression was associated with significant slowing of myocardial
cross-bridge interaction kinetics. For a given level of force (i.e.,
P/P0) after flash photolysis,
the rate of tension development
(kCa) was
significantly depressed in the 9-, 21-, and 33-mo-old rats relative to
the 3-mo-old rats. Likewise,
V0 decreased
significantly in an age-dependent manner, suggesting that the rate of
cross-bridge detachment is also reduced with aging. The age-dependent
decreases in both
kCa and
V0 were well
correlated with increased phenotypic expression of -MHC (Fig.
5, A and
B).
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Factors Underlying Depressed Contractile Properties of Senescent Myocardium
Senescent myocardium is characterized by a significant prolongation of contraction time (5, 6, 10). Contraction duration is influenced at least in part by the time course of SR Ca2+ release and reuptake, which has been a primary focus of age-related research in the heart. Although the rate of Ca2+ release and the amplitude of the Ca2+ transient during a given twitch are similar between young and senescent myocardium (29, 49), the duration of the Ca2+ transient is markedly prolonged in aging (29). This phenomenon may be due to an age-related reduction in the rate of Ca2+ uptake by the SR (11) and/or reduced expression of the SR Ca2+-ATPase (21, 43). In addition, alterations in the level of expression and/or extent of phosphorylation of phospholamban would also be expected to alter the rate of Ca2+ uptake by the SR, since in its nonphosphorylated state phospholamban inhibits the SR Ca2+-ATPase (7). At present, little information is available regarding possible age-related effects on the level of phospholamban expression, but numerous studies have demonstrated that the aged heart is less responsive to-adrenergic
stimulation (19). Although the density of -adrenergic receptors is
unaffected with aging (1), an age-induced depression of
agonist-receptor interaction, G protein function, and/or adenylate
cyclase activation (1, 26) leads to reduced levels of cAMP and
subsequent decreased activation of cAMP-dependent protein kinase (PKA)
(35, 36). The age-related decrease in PKA activity results in reduced
phosphorylation of phospholamban (17) and presumably greater inhibition
of the SR Ca2+ pump.
Although the prolonged duration of contraction has been related to slowed kinetics of intracellular Ca2+ uptake in senescent myocardium, it is likely that slowed kinetics of cross-bridge interaction (i.e., rates of attachment and detachment) also contribute significantly to the overall twitch time course. In striated muscles, Ca2+ binding to troponin C initiates a series of events that permits the strong binding of myosin cross bridges to actin and transition to a force-generating state. During relaxation, the dissociation of Ca2+ from troponin C leads to initiation of inactivation of the thin filament and subsequent detachment of myosin from actin, i.e., transition of cross bridges to non-force-generating states. Although Ca2+ binding to troponin C is required for contraction, complete activation of the thin filament in terms of both tension and the kinetics of tension development appears to arise from synergistic actions of Ca2+ and strong-binding myosin cross bridges (41, 42). In cardiac muscle, there is significant cooperativity of Ca2+ binding to troponin C (44), which is enhanced by strong-binding cross bridges (15, 32, 47). Because of the activating influence of attached cross bridges, the duration of contraction should be influenced by both the kinetics of Ca2+ handling (i.e., the rate of Ca2+ delivery and removal from the myoplasm) and the rates of cross-bridge binding and dissociation.
The kinetics of actin-myosin interaction are believed to be modulated
at least in part by the MHC content. In skeletal muscle fibers, the
maximal rate of tension development is approximately eightfold higher
in fast-twitch fibers expressing type IIb MHC than in slow-twitch
fibers expressing type I MHC, i.e., -MHC (23). Furthermore,
V0 is thought to
be related to MHC isoform content (30). With normal aging, the
transition from juvenile (2-3 mo old) to senescent (33 mo old) is
characterized by an age-dependent shift in the distributions of
ventricular MHC isoforms from ~80% -MHC/20% -MHC at 3 mo to
~30% -MHC/70% -MHC by 33 mo (Fig. 1A). The shift in MHC content in
the present study is similar in magnitude to age-dependent shifts seen
by other investigators (4, 37, 38). The alteration in ventricular MHC
isoform expression occurred independent of any change in ventricular
MLC expression, and we are unaware of any other reports that have shown
any age-dependent shift in the expression of the MLC isoforms in the
rodent ventricle.
Alternatively, modulation of cross-bridge kinetics might arise from
age-related shifts in expression of thin filament proteins, which may
alter thin filament responsiveness to
Ca2+ or strongly bound cross
bridges. Previous work has shown that changes in the relative
distribution of troponin T (16, 27) and tropomyosin isoforms (31) can
alter the Ca2+ sensitivity of
tension. By 2 mo of age, only -cardiac actin is expressed in the
rodent ventricle, and this pattern is maintained into senescence (38).
Similarly, the expressions of the adult cardiac tropomyosin and the
troponin subunit (i.e., troponins T, I, and C) isoforms stabilize by 2 mo of age and are unchanged with normal aging (4). Thus, because the
ventricular MHC isoforms were the only cardiac contractile proteins
that underwent an age-induced alteration in phenotypic expression, we
conclude that the age-related changes in the kinetics of myocardial
cross-bridge interaction are due to altered MHC expression.
In the present study, the kinetics of cross-bridge interaction were
progressively depressed in myocardium as part of the aging process, a
phenomenon which we related to progressive increases in -MHC content
and concomitant decreases in -MHC content. Photolabile caged
Ca2+ compounds, such as
DM-Nitrophen and nitr-7, have been commonly used to examine
cross-bridge interaction kinetics in mammalian striated muscle.
Ca2+ dependence of the rate of
tension development has been observed in skinned preparations from both
adult rabbit psoas muscle (33) and juvenile rat myocardium (3). Similar
results were obtained in the present study, i.e., the rate of tension
development varied with the level of
Ca2+ activation at all ages
examined. Although a Ca2+
dependence of the rate of tension development was consistently observed, myocardium from 9-, 21-, and 33-mo-old rats developed tension
at significantly slower rates at all levels of activation than
myocardium from 3-mo-old rats (Table 3). The age-dependent decline in
the rates of submaximal and maximal tension development was associated
with the age-induced increase in -MHC expression (Fig.
5A). This aging-dependent
relationship of kinetics and -MHC content is consistent with the
approximately threefold greater ATPase activity of -MHC vs. -MHC
(34).
V0 was slowed in
senescent myocardium. Siemankowski et al. (39) previously examined
the rate of ADP dissociation from rabbit skeletal and rat cardiac
muscle actomyosin-S1 preparations and observed muscle-specific (i.e.,
MHC-dependent) differences in ADP dissociation rates. They concluded
that the rate of ADP dissociation from the cross bridge is sufficiently
slow to limit maximal shortening velocity by controlling cross-bridge
detachment from actin. Thus the age-related changes in
V0 observed here
(Fig. 5B) most likely manifest
decreases in the kinetics of cross-bridge detachment due to increased
expression of -MHC. Therefore, it is reasonable to think that a
reduction in the overall rate of cross-bridge transitions leading to
the detachment of myosin from actin slows relaxation and contributes to
an increase in myocardial twitch duration.
In summary, the transition from adulthood to senescence is
characterized by a progressive decline in cardiac function (19). Consistent with previous observations (2), peak LV pressure and
contractility (LV ±dP/dt) were
markedly depressed in senescent animals (Fig. 2,
A and
B). The age-related decline in
myocardial performance undoubtedly arises from multiple variables
acting at various levels of organization within the heart. At the
subcellular level, twitch and Ca2+
handling kinetics are markedly altered, leading to the prolonged contraction time typically seen in senescent rats. At the myofilament level, increased expression of -MHC appears to lead to reduced rates
of tension development and relaxation. Collectively, these factors
likely act in concert to regulate global ventricular function and thus
support the idea that -MHC-induced slowing of the rates of
cross-bridge activation and relaxation contributes to the modulation of
the rate and extent of ventricular pressure development and relaxation
during the isovolumic contraction and relaxation phases of the cardiac cycle.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the expert technical assistance of Dr. James Graham, Larry Whitesell, and Khristen Carlson.
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FOOTNOTES |
|---|
This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-54581 (to R. L. Moss) and American Heart Association, Wisconsin Affiliate, Grant-in-Aid 97-GB-90 (to D. P. Fitzsimons).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. P. Fitzsimons, Dept. of Physiology, Univ. of Wisconsin School of Medicine, Madison, WI 53706 (E-mail: fitzsimons{at}physiology.wisc.edu).
Received 31 August 1998; accepted in final form 13 January 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Abrass, I. B.,
J. L. Davis,
and
P. J. Scarpace.
Isoproterenol responsiveness and myocardial beta-adrenergic receptors in young and old rats.
J. Gerontol.
37:
156-160,
1982.
2.
Anversa, P.,
T. Palackal,
E. H. Sonnenblick,
G. Olivetti,
L. G. Meggs,
and
J. M. Capasso.
Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart.
Circ. Res.
67:
871-885,
1990
3.
Araujo, A.,
and
J. W. Walker.
Kinetics of tension development in skinned cardiac myocytes measured by photorelease.
Am. J. Physiol.
267 (Heart Circ. Physiol. 36):
H1643-H1653,
1994
4.
Ball, K. L.,
and
R. J. Solaro.
Discoordinate regulation of contractile protein gene expression in the senescent rat myocardium.
J. Mol. Cell. Cardiol.
26:
519-525,
1994[Medline].
5.
Besse, S.,
P. Assayag,
C. Delcayre,
F. Carre,
S.-L. Cheav,
Y. Lecarpentier,
and
B. Swynghedauw.
Normal and hypertrophied senescent rat heart: mechanical and molecular characteristics.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H183-H190,
1993
6.
Bhatnagar, G. M.,
G. D. Walford,
E. S. Beard,
S. Humphreys,
and
E. G. Lakatta.
ATPase activity and force production in myofibrils and twitch characteristics in intact muscle from neonatal, adult and senescent myocardium.
J. Mol. Cell. Cardiol.
16:
203-218,
1984[Medline].
7.
Colyer, J.,
and
J. H. Wang.
Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban.
J. Biol. Chem.
266:
17486-17493,
1991
8.
Fabiato, A.
Computer programs for calculating total from specified free and free from specified total ionic concentrations in aqueous solutions containing multiple metals or ligands.
Methods Enzymol.
157:
378-417,
1988[Medline].
9.
Fitzsimons, D. P.,
P. W. Bodell,
and
K. M. Baldwin.
Myocardial functional correlates of cardiac myosin light chain 2 phosphorylation.
J. Appl. Physiol.
68:
2426-2433,
1990
10.
Fraticelli, A.,
R. Josephson,
R. Danziger,
E. Lakatta,
and
H. Spurgeon.
Morphological and contractile characteristics of rat cardiac myocytes from maturation to senescence.
Am. J. Physiol.
257 (Heart Circ. Physiol. 26):
H259-H265,
1989
11.
Froehlich, J. P.,
E. G. Lakatta,
E. Beard,
H. A. Spurgeon,
M. L. Weisfeldt,
and
G. Gerstenblith.
Studies of sarcoplasmic reticulum function and contraction duration in young adult and aged rat myocardium.
J. Mol. Cell. Cardiol.
10:
427-438,
1978[Medline].
12.
Giulian, G. G.,
R. L. Moss,
and
M. L. Greaser.
Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels.
Anal. Biochem.
129:
277-287,
1983[Medline].
13.
Godt, R. E.,
and
B. D. Lindley.
Influence of temperature upon contractile activation and isometric force production in mechanically skinned musle fibers of the frog.
J. Gen. Physiol.
80:
279-297,
1982
14.
Harris, D. E.,
S. S. Work,
R. K. Wright,
N. A. Alpert,
and
D. M. Warshaw.
Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro.
J. Muscle Res. Cell Motil.
15:
11-19,
1994[Medline].
15.
Hofmann, P. A.,
and
F. Fuchs.
Bound calcium and force development in skinned cardiac muscle bundles: effect of sarcomere length.
J. Mol. Cell. Cardiol.
20:
667-677,
1988[Medline].
16.
Hofmann, P. A.,
V. Menon,
and
K. F. Gannaway.
Effects of diabetes on isometric tension as a function of [Ca2+] and pH in rat skinned cardiac myocytes.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H1656-H1663,
1995
17.
Jiang, M. T.,
M. P. Moffat,
and
N. Narayanan.
Age-related alterations in the phosphorylation of sarcoplasmic reticulum and myofibrillar proteins and diminished contractile response to isoproterenol in intact rat ventricle.
Circ. Res.
72:
102-111,
1993
18.
Kaplan, J. H.,
and
C. R. Ellis-Davies.
Photolabile chelators for the rapid photorelease of divalent cations.
Proc. Natl. Acad. Sci. USA
85:
6571-6575,
1988
19.
Lakatta, E. G.
Cardiovascular regulatory mechanisms in advanced age.
Physiol. Rev.
73:
413-467,
1993
20.
Lipman, R. D.,
C. E. Chrisp,
D. G. Hazzard,
and
R. T. Bronson.
Pathologic characterization of brown Norway, brown Norway×Fischer 344, and Fischer 344×brown Norway rats with relation to age.
J. Gerontol.
51:
B54-B59,
1996.
21.
Lompre, A. M.,
F. Lambert,
E. G. Lakatta,
and
K. Schwartz.
Expression of sarcoplasmic reticulum Ca2+-ATPase and calsequestrin genes in rat heart during ontogenic development and aging.
Circ. Res.
69:
1380-1388,
1991
22.
Lompre, A. M.,
B. Nadal-Ginard,
and
V. Mahdavi.
Expression of the cardiac - and -myosin heavy chain genes is developmentally and hormonally regulated.
J. Biol. Chem.
259:
6437-6446,
1984
23.
Metzger, J. M.,
and
R. L. Moss.
Calcium-sensitive cross-bridge transitions in mammalian fast and slow skeletal muscle fibers.
Science
247:
1088-1090,
1990
24.
Moss, R. L.
Ca2+ regulation of mechanical properties of striated muscle.
Circ. Res.
70:
865-884,
1992
25.
Nakao, K.,
W. Minobe,
R. Roden,
M. R. Bristow,
and
L. A. Leinwand.
Myosin heavy chain expression in human heart failure.
J. Clin. Invest.
100:
2362-2370,
1997[Medline].
26.
Narayanan, N.,
and
J. A. Derby.
Alterations in the properties of beta-adrenergic receptors of myocardial membranes in aging: impairments in agonist-receptor interactions and guanine nucleotide regulation accompany diminished catecholamine-responsiveness of adenylate cyclase.
Mech. Ageing Dev.
19:
127-139,
1982[Medline].
27.
Nassar, R.,
N. N. Malouf,
M. B. Kelly,
A. E. Oakeley,
and
P. A. W. Anderson.
Force-pCa relation and troponin T isoforms of rabbit myocardium.
Circ. Res.
69:
1470-1475,
1991
28.
Neely, J. R.,
and
M. J. Rovetto.
Techniques for perfusing isolated rat hearts.
Methods Enzymol.
39:
43-60,
1975[Medline].
29.
Orchard, C. H.,
and
E. G. Lakatta.
Intracellular calcium transients and developed tensions in rat heart muscle. A mechanism for the negative interval-strength relationship.
J. Gen. Physiol.
86:
637-651,
1985
30.
Pagani, E. D.,
and
F. J. Julian.
Rabbit papillary muscle myosin isozymes and the velocity of muscle shortening.
Circ. Res.
54:
586-594,
1984
31.
Palmiter, K. A.,
Y. Kitada,
M. Muthuchamy,
D. F. Wieczorek,
and
R. J. Solaro.
Exchange of - for -tropomyosin in hearts of transgenic mice induces changes in thin filament response to Ca2+, strong cross-bridge binding, and protein phosphorylation.
J. Biol. Chem.
271:
11611-11614,
1996
32.
Pan, B.-S.,
and
R. J. Solaro.
Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers.
J. Biol. Chem.
262:
7839-7849,
1987
33.
Patel, J. R.,
G. M. Diffee,
and
R. L. Moss.
Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.
Biophys. J.
70:
2333-2340,
1996[Medline].
34.
Pope, B.,
J. F. Y. Hoh,
and
A. Weeds.
The ATPase activities of rat cardiac myosin isoenzymes.
FEBS Lett.
118:
205-208,
1980[Medline].
35.
Sakai, M.,
R. S. Danziger,
J. M. Staddon,
E. G. Lakatta,
and
R. G. Hansford.
Decrease with senescence in the norepinephrine-induced phosphorylation of myofilament proteins in isolated rat cardiac myocytes.
J. Mol. Cell. Cardiol.
21:
1327-1336,
1989[Medline].
36.
Sakai, M.,
R. S. Danziger,
R.-P. Xiao,
H. A. Spurgeon,
and
E. G. Lakatta.
Contractile response of individual cardiac myocytes to norepinephrine declines with senescence.
Am. J. Physiol.
262 (Heart Circ. Physiol. 31):
H184-H189,
1992
37.
Schuyler, G. T.,
and
L. R. Yarbrough.
Effects of age on myosin and creatine kinase isoforms in left ventricles of Fischer 344 rats.
Mech. Ageing Dev.
56:
23-28,
1990[Medline].
38.
Schwartz, K.,
L. Carrier,
C. Chassagne,
C. Wisnewsky,
and
K. R. Boheler.
Regulation of myosin heavy chain and actin isogenes during cardiac growth and hypertrophy.
Soc. Exp. Biol.
46:
265-272,
1992.
39.
Siemankowski, R. F.,
M. O. Wiseman,
and
H. D. White.
ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle.
Proc. Natl. Acad. Sci. USA
82:
658-662,
1985
40.
Strang, K. T.,
N. K. Sweitzer,
M. L. Greaser,
and
R. L. Moss.
-Adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats.
Circ. Res.
74:
542-549,
1994
41.
Swartz, D. R.,
and
R. L. Moss.
Influence of a strong-binding myosin analogue on calcium-sensitive mechanical properties of skinned skeletal muscle fibers.
J. Biol. Chem.
267:
20497-20506,
1992
42.
Swartz, D. R.,
R. L. Moss,
and
M. L. Greaser.
Calcium alone does not fully activate the thin filament for S1 binding to rigor myofibrils.
Biophys. J.
71:
1891-1904,
1996[Medline].
43.
Tate, C. A.,
G. E. Taffet,
E. K. Hudson,
S. L. Blaylock,
R. P. McBride,
and
L. H. Michael.
Enhanced calcium uptake of cardiac sarcoplasmic reticulum in exercise-trained old rats.
Am. J. Physiol.
258 (Heart Circ. Physiol. 27):
H431-H435,
1990
44.
Tobacman, L. S.
Thin filament-mediated regulation of cardiac contraction.
Annu. Rev. Physiol.
58:
447-481,
1996[Medline].
45.
VanBuren, P.,
D. E. Harris,
N. A. Alpert,
and
D. M. Warshaw.
Cardiac V1 and V3 myosins differ in their hydrolytic and mechanical activities in vitro.
Circ. Res.
77:
439-444,
1995
46.
Wallenstein, S.,
C. L. Zucker,
and
J. L. Fleiss.
Some statistical methods useful in circulation research.
Circ. Res.
47:
1-9,
1980
47.
Wang, Y.-P.,
and
F. Fuchs.
Length, force, and Ca2+-troponin C affinity in cardiac and slow skeletal muscle.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1077-C1082,
1994
48.
Wolff, M. R.,
P. P. de Tombe,
Y. Harasawa,
D. Burkhoff,
S. Bier,
W. C. Hunter,
G. Gerstenblith,
and
D. A. Kass.
Alterations in left ventricular mechanics, energetics and contractile reserve in experimental heart failure.
Circ. Res.
70:
516-529,
1992
49.
Xiao, R.-P.,
H. A. Spurgeon,
F. O'Conner,
and
E. G. Lakatta.
Age-associated changes in beta-adrenergic modulation on rat cardiac excitation-contraction coupling.
J. Clin. Invest.
94:
2051-2059,
1994.
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) and 33-mo-old (
) rats. * Statistical difference
3 vs. 33 mo, P < 0.05.
), 5.42 ± 0.03; 21 mo (
),
5.56 ± 0.03; 33 mo (
), 5.52 ± 0.02 pCa units. Cell
characteristics are listed in Table 
)
rates of tension development are plotted as a function of percent