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1 Department of Pharmaceutical Sciences, 2 Department of Pharmacology and Toxicology, and 3 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The
effect of aging on cardiac membrane currents remains unclear. This
study examined the inward rectifier K+ current
(IK1), the transient outward K+
current (Ito), and the L-type
Ca2+ channel current (ICa,L)
in ventricular myocytes isolated from young adult (6 mo) and aged (>27
mo) Fischer 344 rats using whole cell patch-clamp techniques. Along
with an increase in the cell size and membrane capacitance, aged
myocytes had the same magnitude of peak IK1 with
a greater slope conductance but displayed smaller steady-state
IK1. Aged myocytes also had a greater
Ito with an increased rate of activation, but the
Ito inactivation kinetics, steady-state
inactivation, and responsiveness to L-phenylephrine, an
1-adrenergic agonist, were unaltered. The magnitude of
peak ICa,L in aged myocytes was decreased and
accompanied by a slower inactivation, but the
ICa,L steady-state inactivation was unaltered. Action potential duration in aged myocytes was prolonged only at 90%
of full repolarization (APD90) when compared with the
action potential duration of young adult myocytes. Aged myocytes from Long-Evans rats showed similar changes in Ito
and ICa,L but an increased
IK1. These results demonstrate aging-associated
changes in action potential, in morphology, and in
IK1, Ito, and
ICa,L of rat ventricular myocytes that possibly
contribute to the decreased cardiac function of aged hearts.
ion channels; action potential; patch clamp
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NUMEROUS STUDIES in humans and animals have shown a significant loss of ventricular myocytes and reactive hypertrophy of the remaining myocytes in normal aged hearts (2, 11, 24, 25). These aging-associated anatomical changes, which could result from both apoptosis and necrosis (16), are associated with the development of ventricular dysfunction and a decrease in the capacity of the heart to respond to neurohumoral control and inotropic intervention (1, 21). The decreased ventricular function and reserve capacity in senescent hearts are also associated with electrophysiological alterations, including changes in action potential (AP) configuration (9, 28), prolongation of atrioventricular conduction (6, 11, 27, 29), and decreases in heart rate (27, 29). However, the observed aging-associated changes in the AP and underling membrane currents are still uncertain and somewhat contradictory. A study (9) using 4-aminopyridine (4-AP), an inhibitor of the transient outward K+ current (Ito), suggested that the change in AP configuration in aged human atrial fibers is due, in part, to an increase in Ito, although no membrane current was measured in that study. In contrast, measurement of transmembrane AP in right ventricular papillary muscle and in single rat ventricular myocytes showed a prolongation of AP duration in aged rats (30, 31). Studies of whole cell currents in rats suggested an aging-related reduction of Ito (30) with no changes in L-type Ca2+ current (ICa,L) (30, 32). Moreover, a study of Na+-dependent Ca2+ influx in sarcolemmal membrane vesicles showed a decrease in Na+/Ca2+ exchange in the aged myocardium (13), and Kennedy et al. (18) have shown a decrease in Na+/K+ pump current of ventricular myocytes isolated from aged rats. All these changes could affect the cardiac AP and contraction. However, taken together, these changes could not account for the decreased cardiac contractile performance in aged hearts. Thus the mechanism underlying the aging-related changes in AP configuration and heart rate remains unclear.
In the present study, we reexamined the effect of aging on whole cell membrane currents in ventricular myocytes isolated from young adult and aged rats using conventional patch-clamp techniques in combination with square voltage pulses and AP waveforms. Our results show that in addition to cellular hypertrophy, aged ventricular myocytes have significantly greater Ito and smaller ICa,L in both Fischer 344 (F344) and Long-Evans (L-E) rats. The changes in Ito and ICa,L, accompanied by alterations in their kinetics and gating properties, could account for the aging-associated changes in AP configuration, contractile function, and heart rate. A preliminary report of some of these results has been presented in abstract form (23).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
All experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Male young adult (6 mo of age, 280-300 g body wt) and aged (27-28 mo, 380-425 g body wt) F344 rats were purchased from the National Institutes of Health. All animals were maintained on a 12:12 h light-dark cycle and were allowed free access to food and water for at least 2 mo before being used in this study. Some experiments used male L-E rats, 29-36 mo of age (aged, 300-510 g body wt), which were used previously for behavioral studies by another investigator at the University of Arkansas for Medical Sciences. These aged and the corresponding young adult L-E rats (3-4 mo of age, 230-340 g body wt) were purchased from Harlan Sprague Dawley (Indianapolis, IN).Myocyte Isolation
Single adult ventricular myocytes were isolated from the hearts of young adult and aged rats using protocols described previously (22). Briefly, hearts were rapidly excised and perfused at 37°C via the aorta with an oxygenated control buffer solution, followed by a 5-min perfusion with Ca2+-free buffer solution. The control buffer solution contained (in mM) 110 NaCl, 3.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 0.2 CaCl2, and 11 glucose (pH 7.4 in 95% O2-5% CO2 at 37°C). Hearts were then perfused for 20 min with a buffer solution containing 25 µM CaCl2 plus 0.5 mg/ml collagenase. The ventricles were removed, minced, rinsed with control buffer solution, and shaken in a water bath at 37°C for 2-3 periods of 10 min each. Isolated ventricular myocytes were then plated into 60-mm culture dishes (Falcon) containing antibiotic-free, bicarbonate-buffered culture medium 199 (60%, GIBCO, Grand Island, NY) with 36% Earle's balanced salt solution composed of (mM) 116 NaCl, 4.7 KCl, 0.9 NaH2PO4, 0.8 MgSO4, 26 NaHCO3, and 5.6 glucose and 4% fetal bovine serum (GIBCO) (pH 7.4 in 5% CO2-95% air at 37°C). Rod-shaped myocytes were used after 24-48 h in culture. Note that there was no significant difference in the current density of K+ and Ca2+ currents in cells maintained in culture up to 72 h. However, for the sake of simplicity, all data regarding kinetics were obtained from myocytes in culture for 24 h.Electrophysiological Measurements
Ventricular myocytes were placed on the heated stage of an inverted microscope (Nikon Diaphot, Irving, TX) and perfused with a normal Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 0.8 MgCl2, 10 HEPES/Tris, and 5.6 glucose (pH of 7.40 at 37°C; 290 mosM). Cells were patch clamped in the whole cell configuration by conventional techniques (12) using a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA) as previously described (22). Briefly, patch electrodes were filled with a pipette solution and had a tip resistance of 1-4 M
. Recorded currents
were filtered at 2-5 kHz through a 4-pole low-pass Bessel filter
and sampled at 5-16.7 kHz with a PC computer using pCLAMP 6.03 software through a Digidata 2000A acquisition system (Axon Instruments). Series resistance (Rs) was
compensated between 90 and 95% so that peak currents could be clearly
separated from the capacity current transient. Under these conditions,
Rs was <1 M, and the estimated maximal error
was <5 mV for a maximal Ito (measured at +60
mV) of 5 nA. All measured membrane currents were normalized to membrane
capacitance (Cm); the capacity current transient
recorded in response to a 5-mV hyperpolarizing pulse was integrated and
divided by the given voltage to give total Cm
for each cell. The measurements of the inward rectifier K+
current (IK1) and Ito,
which used the same pipette solution, were carried out at room
temperature; ICa,L and AP were recorded at
37°C.
To measure whole cell IK1, myocytes were
perfused with the normal Tyrode solution and voltage clamped at 
40 mV
to inactivate Na+ channels. Myocytes were internally
dialyzed with K+-containing buffer solutions consisting of
(in mM) 120 K-aspartate, 25 KCl, 2 MgCl2, 0.5 CaCl2, 1 EGTA, 5 Na2ATP, 0.4 Li4GTP, 10 HEPES/Tris, and 5.6 glucose (pH 7.20 at room
temperature). The concentration of EGTA used in these experiments was
designed to prevent cell contraction and the possible effects of cell
movement on the AP and current measurements. The current-voltage
(I-V) curve was obtained by applying 550-ms
voltage pulses between 110 and +60 mV from the holding potential at
0.2 Hz. The magnitude of peak IK1 was analyzed 3 ms after the beginning of the pulses, when the capacity transient was
completed and could be separated. Steady-state IK1 was obtained by averaging the current level
during 5 ms at the end of the pulses between 90 and 110 mV.
To isolate Ito, myocytes were voltage clamped at
60 mV and externally perfused with a Na+-free solution
consisting of (in mM) 140 N-methyl-D-glucamine (NMDG)-Cl, 5.4 KCl, 0.8 MgCl2, 0.5 BaCl2, 0.2 CdCl2, and 10 HEPES/Tris base (pH 7.40 at room
temperature). To minimize the influence of IK1
on determining the activation of Ito,
Ba2+ was used to completely block
IK1. Note that Ba2+ (0.5 mM) also
slightly decreased Ito (~15%) in both aged
and young ventricular myocytes. Na+-associated currents
were suppressed by the use of Na+-free, NMDG-containing
solutions. ICa,L was blocked by 0.2 mM Cd2+, and the membrane current associated with
Na+/Ca2+ exchange was eliminated by the absence
of external Na+ and Ca2+. Under these
conditions, Ito could be clearly analyzed.
Ito was elicited by 20-ms or 1-s voltage pulses
between 80 and +60 mV in 10-mV increments from a holding potential of
60 mV at 0.33 and 0.1 Hz, respectively. The kinetics of
Ito activation were described by the product of
a rising and falling function according to the Hodgkin-Huxley model
(14), as defined by the following equation:
It = A · [1 exp(t/
A)]p · exp(t/I) + B, where
A and I are time constants of activation and inactivation, respectively; the power, p, is set to 3 to obtain the
best fit for Ito (4); A
is an amplitude scalar, and B is the steady-state current
level. The steady-state inactivation relationship was determined using
a double-pulse protocol; a 1-s prepulse to potentials between 110 and
+60 mV was followed by a fixed 975-ms test pulse to +60 mV. The
difference current between the peak and steady-state levels during the
test pulse was used to obtain inactivation parameters. Steady-state
activation was determined by the conductance-voltage relationship of
Ito obtained from currents in response to the
1-s prepulse described above. The conductance (G) was
calculated using the following equation: G = I / (V Erev),
where Erev is the reversal potential for
Ito (approximately 75 mV estimated by tail
currents obtained from 20-ms pulse protocols used for examining
activation of Ito described above). In some
experiments, steady-state activation was also determined using a
double-pulse protocol; a 15-ms prepulse to potentials between 50 and
+60 mV from the holding potential of 60 mV was followed by a fixed
80-ms test pulse to 20 mV. The tail Ito in
response to the test pulse reflects the instantaneous conductance of
Ito activated at prepulse potentials
(4). The voltage dependence of steady-state activation was
obtained by plotting relative conductance
(G/Gmax or tail
Ito /maximal tail Ito) as
a function of the prepulse potential. Both protocols gave similar
results for the parameters of steady-state activation of
Ito. To analyze more accurately the kinetics of
Ito, experiments were performed at room
temperature to slow down the kinetics. Data obtained for steady state
and kinetics of inactivation and activation were curve-fit by the
Boltzmann distribution and a biexponential decay, respectively, using
the Marquardt-Levenberg nonlinear least-squares curve fitting algorithm
included in Origin (Microcal Software, Northampton, MA).
Whole cell ICa,L was measured as described
previously (22). Briefly, myocytes were perfused with
normal Tyrode solution, followed by an external solution consisting of
(in mM) 140 NMDG-Cl, 0.8 MgCl2, 2 CaCl2, 2 4-AP, and 10 HEPES/Tris base (pH adjusted to 7.40 at 37°C). The
pipette solution contained (in mM) 100 CsOH, 70 aspartic acid, 11 CsCl,
15 tetraethylammonium (TEA) chloride, 2 MgCl2, 5 MgATP, 10 EGTA, 0.1 CaCl2, 5 pyruvic acid, 5.6 glucose, 5 Tris2-phosphocreatine, 0.4 Li4GTP, and 10 HEPES/Tris base (pH 7.20 at 37°C). ICa,L was
measured 1-2 min after the patched membrane was ruptured in normal
Tyrode solution; this current represented ICa,L
in near physiological solutions. The biophysical properties of
ICa,L were then analyzed in NMDG-containing
solutions to efficiently isolate ICa,L from the
contamination of other currents; under these conditions K+
currents were suppressed by internal Cs+ and
TEA+ as well as by external K+-free solutions
containing 4-AP. Na+ currents were suppressed by the use of
Na+-free NMDG-containing solutions. The I-V
relationship of ICa,L was obtained by giving
250-ms voltage pulses to potentials between 60 and +60 mV from a
holding potential of 40 mV (in normal Tyrode solutions) or 70 mV
(in NMDG solutions) in 10-mV increments at 0.2 Hz. Steady-state
inactivation and activation relationships were determined using a
gapped double-pulse protocol once every 2 s; a 250-ms prepulse to
potentials between 90 and +50 mV was followed by a 10-ms return to
the holding potential and then a fixed 250-ms test pulse to +10 mV.
AP and Membrane Currents Measured Using AP Waveforms
Measurements of AP and membrane currents elicited by AP clamp were performed in myocytes that were internally dialyzed with K+-containing solution for IK1 and externally perfused with Tyrode solutions at 37°C. The AP was elicited with a 0.5-ms depolarizing pulse at 0.5 Hz using a current-clamp mode. The AP duration (APD) from an average of 3 to 5 consecutive AP was analyzed at 20, 50, 75, and 90% of full repolarization (APD20, APD50, APD75, and APD90, respectively). After AP was measured the recording was switched to the voltage-clamp mode. Membrane currents of the same cell were elicited using its AP waveform as the command voltage. In some experiments, the AP waveform used was obtained from the averaged AP of young adult cells. Currents from the same cells were also examined using conventional step-voltage pulses at 0.2 Hz. Cd2+- and 4-AP-sensitive currents were obtained by subtracting the current in the presence of Cd2+ or 4-AP, respectively, from the current before application of each chemical.Chemicals
Most reagents were purchased from Sigma Chemical (St. Louis, MO) and were directly added when needed. Stock solutions of l-phenylephrine (PE) and nadolol (10
1 M) were
prepared in Milli-Q water and ethanol, respectively. The final
concentration of ethanol in extracellular solutions was less than
0.01% and had no effect on membrane currents (data not shown). After
addition of these chemicals, the pH of the solutions was readjusted as necessary.
Statistics
Values are presented as means ± SE. Statistical significance was evaluated by the two-tailed Student's t-test or, when more than two conditions were compared, by one-way analysis of variance (ANOVA) with Duncan's multiple range test. Differences with P < 0.05 were considered significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Aging-Associated Alterations in Cell Size and Cm of Ventricular Myocytes
Table 1 shows the comparison of cell dimension and Cm in ventricular myocytes isolated from aged and young adult, F344 and L-E rat hearts. Both cell length and width were significantly greater in aged myocytes of both strains. The ratio of length to width in young adult myocytes of both strains was similar (5.5 in F344 versus 5.4 in L-E rats). However, this ratio was decreased in aged F344 (to 5.2), whereas it was increased in aged L-E rats (to 5.8). Although the type of cell hypertrophy was different in the two strains, the aging-associated changes in cell size were consistent with findings reported previously by other investigators (10). Additionally, the measured Cm of aged myocytes was 23.1% and 15.7% greater than that of young adult myocytes in F344 and L-E rats, respectively. These results showing aging-induced cardiac cellular hypertrophy in male rats are consistent with findings reported for humans (24).
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Aging-Associated Changes in Whole Cell Membrane Currents
Myocytes were internally dialyzed with K+- and Na+-containing buffer solutions and externally perfused with normal Tyrode solution. Figure 1 shows the I-V relationships of membrane currents in aged and young adult F344 myocytes elicited by 550-ms voltage pulses to potentials between110 and +60 mV from a holding potential of 40
mV. The comparison of IK1 (observed in the range
between 90 and 110 mV and completely blocked by 0.5 mM
Ba2+) between aged and young adult F344 ventricular
myocytes was summarized in Table 2. The
peak IK1 was comparable in the two aged groups. However, steady-state IK1 in aged F344 myocytes
was significantly smaller than that in young myocytes (Table 2).
Representative traces of IK1 recorded from aged
and young myocytes are shown in Fig. 1 (inset). Table 2 also
shows that there were no differences in the peak and steady-state
levels in young adult F344 and L-E ventricular myocytes. However, in
L-E rats both peak and steady-state IK1 of aged
myocytes were significantly greater than those in L-E young adult
cells.
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The activation of IK1 elicited by
hyperpolarizing pulses to potentials more negative than 90 mV was
followed by an inactivation, most of which could be best fit with two
exponentials (3). Table 3
shows that the fast time constants (f) did not differ between aged and young adult myocytes, but the slow time constants (s) of IK1 inactivation was
significantly increased in aged F344 myocytes, possibly because of the
low level of steady-state IK1. In contrast, the
IK1 in L-E aged myocytes appeared to inactivate more rapidly than that in young adult myocytes (Table 3). The s of IK1 inactivation did not
differ between the two age groups of L-E rats (aged, 146.6 ± 28.8 ms, n = 8 versus young adult, 145.1 ± 8.7 ms,
n = 8 at 110 mV). The slope conductances of peak IK1 in aged and young F344 myocytes were 0.64 and 0.61 nS/pF, respectively. When the average
Cm was taken into account, the equivalent
conductances were 126 nS (aged) and 97 nS (young). The slope
conductance in aged and young L-E myocytes was 0.82 and 0.66 nS/pF,
respectively, and the equivalent conductance was 145 nS (aged) and 101 nS (young). These results show an aging-associated increase in the
conductance of IK1 in both strains of rats.
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Results in Fig. 1 also show that in aged myocytes peak
ICa,L tended to be smaller, whereas the peak
outward current (measured at +60 mV) tended to be greater when compared
with young adult myocytes. These aging-related trends were not
statistically significant, because these two currents were not isolated
from other membrane currents and voltage clamped at 40 mV.
Aging-related alterations in both peak ICa,L and
Ito were verified in the following experiments that isolated each current from other cation currents.
Aging-Associated Changes in Ito
The magnitude of Ito.
Figure 2A shows the
I-V relationships of Ito in aged and
young myocytes from F344 rats measured as both peak and net active (i.e., the difference between peak and steady-state level at the end of
a 1-s test pulse) currents in Na+- and
Ca2+-free solutions containing 0.2 mM Cd2+ and
0.5 mM Ba2+. Results show that peak and net
Ito (measured at +60 mV) in aged myocytes were
39% and 46% greater than those in young adult myocytes, respectively
(i.e., peak: 27.2 ± 2.5 pA/pF, n = 9 in aged vs. 19.6 ± 1.5 pA/pF, n = 25 in young adult; net:
21.1 ± 2.3 pA/pF, in aged vs. 14.4 ± 1.3 pA/pF in young
adult, P < 0.01). Representative superimposed current
traces of Ito family in aged and young adult myocytes are shown in Fig. 2, B and C,
respectively. Figure 2A (inset) shows that peak
Ito measured at +60 mV in aged myocytes elicited
by 20-ms voltage pulses was also consistently 40% greater than that in
young adult cells (aged: 32.4 ± 1.8 pA/pF, n = 10 vs. young adult: 23.2 ± 1.8 pA/pF, n = 25). With
the use of a 1-s pulse protocol, the steady-state level after the
inactivation of Ito (i.e., at the end of the 1-s
test pulse) was comparable in the two age groups (6.1 ± 1.8 pA/pF
in aged vs. 5.2 ± 0.4 pA/pF in young measured at +60 mV). Similar
results were found in L-E rats, where aged myocytes had significantly
greater peak and net active Ito measured between
+10 and +60 mV than that of young adult myocytes (e.g., peak at +60 mV:
20.8 ± 1.3 pA/pF, n = 6, in aged vs. 12.7 ± 1.3 pA/pF, n = 8, in young adult, P < 0.01; net: 16.9 ± 1.1 pA/pF in aged vs. 9.7 ± 1.4 pA/pF in
young adult, P < 0.01). The difference in peak
Ito measured at +60 mV during 20-ms voltage
pulses between aged and young myocytes was more profound (32.6 ± 3.5 pA/pF in aged, n = 6, vs. 21.7 ± 3.0 pA/pF in
young adult, n = 10, P < 0.05).
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1-Adrenergic responsiveness of Ito.
Figure 3 shows the
1-adrenergic responsiveness of
Ito in aged and young adult myocytes from L-E
rats. Exposure for 7-10 min to 30 µM PE in the presence of 1 µM nadolol (a 
-adrenergic antagonist) significantly reduced peak
Ito measured at +60 mV by 12.4 ± 2.2% (n = 4, P < 0.05, paired
t-test, Fig. 3A) in aged ventricular myocytes and by
13.6 ± 0.8% (n = 6, P < 0.05, paired t-test, Fig. 3B) in young myocytes; PE
also decreased the net active Ito by 11.2 ± 1.0% (n = 4) and 11.9 ± 1.1%
(n = 6) in aged and young L-E myocytes, respectively.
Similarly, 30 µM PE decreased peak Ito
(measured at +60 mV) of aged and young F344 myocytes by 13.9 ± 0.5% (n = 3) and 12.9 ± 2.0% (n = 4), respectively. These data suggest that the aging process did not
alter the 1-adrenergic responsiveness of
Ito in rat ventricular myocytes.
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The kinetics of Ito.
After initial rapid activation (e.g., in Fig. 2B),
Ito was inactivated in a biexponential manner
when measured between +10 and +60 mV. Data were curve fit using a
double exponential function to obtain the f and
s of inactivation. The two time constants and their
voltage dependence were not different in aged and young F344 myocytes
over the range between +10 and +60 mV (e.g., f of
Ito inactivation at +60 mV: 40.3 ± 2.8 ms
in 7 aged vs. 40.7 ± 1.3 ms in 16 young adult; s:
486 ± 72 ms in aged and 514 ± 41 ms in young
adult). Similar findings were obtained from L-E rats
(f measured at +60 mV: 40.5 ± 0.7 ms in 7 aged vs.
39.3 ± 2.6 ms in 10 young adult; s: 496 ± 41 ms in aged vs. 414 ± 72 ms in young adult). Thus results
demonstrated that senescence has no effect on the time constants of
Ito inactivation or their voltage dependence.
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Steady-state inactivation of Ito.
Steady-state inactivation of Ito was obtained
using a double-pulse protocol (see METHODS). Figure
5A shows that data were fit by
a Boltzmann equation, I/Imax = 1/{1 + exp[(V V1/2)/k]}, where
I/Imax is the ratio of current to
maximum current, V1/2 is the half-maximum
inactivation potential and k is the slope factor. Table
4 shows that there was no significant
difference in the steady-state inactivation of
Ito in the two age groups of F344 or L-E rats.
Figure 5A also shows that in the presence of 30 µM PE,
V1/2 in aged L-E myocytes was shifted to
34.9 ± 0.7 mV with no change in k (5.5 ± 0.4 mV, n = 6), whereas V1/2 in
young adult L-E myocytes was shifted to 35.7 ± 0.8 mV with a
slight increase in k (6.4 ± 0.5 mV, n = 6). Similar results showing a PE-induced negative shift in
V1/2 were observed in F344 rats (data not
shown). Interestingly, data obtained from both aged and young myocytes
in the presence of PE were better described by the sum of two Boltzmann
distributions (Fig. 5A). The major component, which was
~87% in both aged and young myocytes, had V1/2 values of 33.6 mV and 34.7 mV,
respectively, and k values of 4.3 and 4.1 mV in aged and
young adult cells, respectively. These values were similar to those
estimated by a single Boltzmann distribution. Nevertheless, the data
show that steady-state inactivation of Ito and
the PE-induced shift in V1/2 (approximately 5
mV) are not altered during aging, reinforcing the notion that aging does not alter the 1-adrenergic responsiveness of
Ito (see Fig. 3).
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Steady-state activation of Ito.
Figure 5B shows the steady-state activation of
Ito in both aged and young adult myocytes by
plotting normalized conductance as a function of prepulse potential
(see methods). Data were fit by a Boltzmann equation,
G/Gmax = 1/{1 + exp[(V1/2 V)/k]} to give V1/2
(half-maximum activation potential) and k. The steady-state activation of Ito was not different between the
two age groups in F344 and L-E rats (see Table 4). Figure 5B
also shows that when L-E cells were exposed to 30 µM PE,
V1/2 in both groups shifted to more negative
potentials (6.0 ± 0.8 mV, n = 6 in aged and
5.3 ± 1.1 mV, n = 4 in young myocytes) without
significant changes in k (12.0 ± 0.4 mV in aged and
12.2 ± 0.6 mV in young myocytes). A PE-induced negative shift in
V1/2 was also observed in F344 rats (data not
shown). These results suggest that senescence has little effect on the
steady-state activation of Ito or its response to PE.
Aging-Associated Changes in ICa,L
The magnitude of ICa,L.
Figure 6 shows the
I-V relationships of ICa,L
in aged and young adult F344 ventricular myocytes internally dialyzed
with K+- and Na+-free buffer solutions and
externally perfused with normal Tyrode solution. Myocytes were held at
40 mV; peak ICa,L (measured at 0 mV) of aged
F344 myocytes was 14% smaller than that of young adult F344 myocytes
(12.7 ± 0.6 pA/pF, n = 16 in aged versus 14.7 ± 0.7 pA/pF, n = 27 in young adult;
P < 0.05). Similarly, peak
ICa,L (measured at 0 mV) in aged L-E myocytes
was 15% smaller than that in young adult myocytes (12.4 ± 1.3 pA/pF, n = 35 in aged versus 14.6 ± 1.5 pA/pF,
n = 21 in young adult; P < 0.02). The
difference in the magnitude of peak ICa,L
between the two groups was larger in the negative than in the positive
potential limb, resulting in a slight aging-associated shift of the
I-V curve to more positive potentials.
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Kinetics of ICa,L.
After its rapid initial activation, ICa,L
inactivates in a biexponential manner. Figure
7A shows the two time
constants of peak ICa,L inactivation (measured
at 0 mV) in aged and young adult F344 ventricular myocytes in normal
Tyrode solution. The s of ICa,L
inactivation was increased in aged compared with young adult myocytes,
whereas there was no difference in the f in the two groups. Figure 7B shows that a similar aging-associated
increase in the s of ICa,L was
observed in L-E ventricular myocytes, whereas the f was
also comparable in the two age groups. Figure 7C
demonstrates this senescence-related decrease in
ICa,L inactivation by superimposing current
traces of ICa,L obtained from aged and young
adult myocytes when normalized to peak magnitude. When
ICa,L was isolated in K+- and
Na+-free solutions (NMDG substitute) containing 2 mM
Ca2+, the aging-related increase in the s of
ICa,L inactivation was also observed (aged:
23.1 ± 1.0 ms, n = 19 vs. young adult: 18.0 ± 1.6 ms, n = 4; P < 0.02). Under
this condition, the f remained comparable in the two age
groups (aged: 2.8 ± 0.1 ms vs. young adult: 2.9 ± 0.3 ms).
These data demonstrate an aging-associated decrease in the slow phase
of ICa,L inactivation.
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Steady-state inactivation and activation of ICa,L.
Steady-state inactivation and activation of
ICa,L were examined while myocytes were voltage
clamped at 70 mV and perfused in NMDG solution containing 2 mM
Ca2+. Data were curve fit by a Boltzmann equation to obtain
V1/2 and k. Figure
8A shows that there was no
significant difference in the steady-state inactivation of
ICa,L in the two age groups (see also Table
5). In contrast, the
V1/2 of steady-state activation of
ICa,L in aged myocytes appeared to shift to more
positive potentials compared with young myocytes, with no significant
change in k. The shift in V1/2 of
steady-state activation (approximately +3 mV) might account for the
reduced peak ICa,L and the shift in the
I-V curve to more positive potentials in aged myocytes.
|
|
Recovery from inactivation of ICa,L.
Figure 8B shows the time course of recovery of
ICa,L from inactivation using a double-pulse
protocol. Superimposed current traces are shown in Fig. 8B
(inset). Recovery from inactivation was voltage dependent
with ICa,L recovering more rapidly at more negative potentials. When myocytes were voltage clamped at 70 mV, the
recovery process was described by a single exponential function with
time constants of 30.3 ± 3.1 ms (n = 8) and
27.8 ± 3.6 ms (n = 3) in aged and young adult
myocytes, respectively. When cells were held at 40 mV, recovery from
ICa,L inactivation tended to be more rapid in
aged compared with young adult myocytes (time constant: 84.4 ± 9.2 ms, n = 8 in aged versus 97.3 ± 16.0 ms,
n = 3 in young adult); however, this difference was not
statistically significant. Thus these results show that aging has no
effect on the recovery of ICa,L from inactivation.
AP and Membrane Currents Elicited by AP Waveforms in Aged and Young Adult Myocytes
The AP of ventricular myocytes in the two age groups was monitored in the current-clamp mode of the whole cell configuration when F344 myocytes were perfused in normal Tyrode solution. The measured parameters of elicited AP in aged and young ventricular myocytes from F344 rats were summarized in Table 6. F344 aged myocytes displayed a prolong APD only at APD90 and a shorter amplitude of AP with no significant change in resting membrane potential when comparing to young adult myocytes. Figure 9, A and B, shows representative APs measured from an aged and a young F344 myocyte, respectively. The APD20 to APD75 of aged myocytes was slightly shorter than that of young myocytes, but the APD90 was longer than that of young myocytes. Exposure of myocytes to 3 mM 4-AP caused a prolongation of APD50 to 342 ± 36% (in aged, n = 7) and 291 ± 28% (in young, n = 10) of control values.
|
|
Figure 9 also shows the membrane currents of myocytes in these two
cells elicited using their own digitalized AP waveforms in the same
conditions used for obtaining the AP. Figure 9, C and
D, shows the membrane current in aged and young adult F344 myocytes, respectively, before and during exposure to 0.2 mM
Cd2+. Subtracting the currents in the presence of
Cd2+ from the control gave a Cd2+-sensitive
current as shown in Fig. 9, E and F. Although
Cd2+ may have effects on currents other than
ICa,L, the Cd2+-sensitive currents
of 17.0 ± 1.4 pA/pF (n = 19) and 21.6 ± 2.6 pA/pF (n = 9) in aged and young myocytes,
respectively, are consistent with ICa,L in two
age groups. The difference in Cd2+-sensitive currents
between the two age groups was similar to that observed in
ICa,L when elicited using rectangular voltage pulses to +60 mV from a holding potential of 40 mV (aged: 10.3 ± 0.4 pA/pF, n = 20; young adult: 12.0 ± 0.7 pA/pF, n = 15; P < 0.05) under the
same conditions using pipette and perfusion solutions containing normal
Na+ and K+. Similarly, the 4-AP-sensitive
current in aged and young adult myocytes were obtained as the
difference between currents recorded before and during exposure to 3 mM
4-AP. The 4-AP-sensitive currents in aged myocytes was greater than
those in young adult (8.1 ± 1.0 pA/pF, n = 6 in
aged vs. 5.9 ± 0.5 pA/pF, n = 9 in young adult; P < 0.05), which is consistent with the aging-related
increase in isolated Ito elicited with step
voltage pulses (shown in Fig. 2). These data demonstrate that different
approaches gave comparable results, i.e., that aged myocytes have a
smaller ICa,L and a greater Ito than young adult myocytes.
| |
DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
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Aging-associated cardiac electrophysiological alterations include changes in AP configuration (5, 9, 28, 30, 31), prolongation of atrioventricular conduction (6, 11, 27, 29), and decreases in heart rate (27, 29). Kennedy et al. (18) have shown a decrease in Na+/K+ pump current in aged ventricular myocytes from L-E rats; however, this change cannot account for all the aging-related changes observed in AP configuration and heart rate. In the present study, we examined whether aging affects IK1, Ito, and ICa,L, the three major determinants of AP configuration in rat ventricular myocytes. We found that aging is associated with 1) an increase in Ito that is coupled with faster activation; 2) a decrease in ICa,L with a slower inactivation and a positive shift in steady-state activation; and 3) a change in IK1. Aging-associated changes in Ito and ICa,L are identical in both F344 and L-E rats, and (4) there was a prolongation of AP only at APD90.
Because peak and steady-state IK1 in young adult
myocytes did not differ between F344 and L-E rats, the effect of aging
on IK1 seems to be strain specific. Peak
IK1 of ventricular myocytes was not different in
aged and young adult F344 rats, whereas it was greater in aged L-E
rats. The inactivation of peak IK1 to steady-state levels also appears to be altered in aged ventricular myocytes. The ratios of steady-state current to peak
IK1 (at 110 mV), indicating the degree of
inactivation (3), for aged and young myocytes were 0.51 and 0.67, respectively, in F344 rats and 0.74 and 0.64, respectively,
in L-E rats. The greater degree of inactivation of
IK1 in aged F344 myocytes is associated with a
slower slow component of inactivation, whereas the smaller degree of
inactivation of IK1 in aged L-E myocytes is
accompanied by a more rapid fast component. Further studies are
required to determine whether the greater slope conductance of
IK1 observed in aged L-E rats indicates
different subunits of the IK1 family or more open channels and determine the mechanisms by which aging alters the
inactivation of IK1 in the two strains.
The present study also demonstrates an increase in
Ito in aged ventricular myocytes of both strains
(by ~40% in F344 rats and >50% in L-E rats). Because the magnitude
of Ito of young myocytes in the two strains is
comparable, the greater increase in Ito in aged
L-E myocytes could be attributed to the fact that the senescent L-E
rats were older at the time of euthanization (average age: 31.4 mo old)
compared with the aged F344 rats (average age: 26.8 mo). An age-related
increase in the current density of Ito has also
been reported to occur when compared with atrial myocytes obtained from
young (1 day-2.5 yr) and adult (11-68 yr) humans; however, adult
and aged myocytes were not compared in that study (7). In
contrast to our findings, a study (30) using ventricular myocytes of Wistar rats showed an aging-related decrease in
Ito. The discrepancy between these two studies
might be attributable to different experimental conditions and/or the
strains of rats used. For example, data presented in the present study
were obtained from myocytes in primary culture, whereas others used
freshly isolated myocytes. The current density of
Ito in the two aged groups is stable in culture
for 48 h. Second, the Ito of Wistar myocytes was measured using pipette solutions containing 156 mM Cl, which may affect the accuracy of the
Ito measurement because an outward
ICl is activated in the same voltage range
(i.e., between 0 and +60 mV). In addition, the effect of
Cl on Ito may vary in different
age groups; this possibility was suggested by results from the study
(30) using Wistar rats, which showed greater residual
currents in aged myocytes in the presence of 4-AP. Moreover, the
previous study (30) defined the magnitude of
Ito as the difference between peak outward
current and the level at the end of 200-ms voltage pulses, a time at
which Ito did not reach steady-state levels.
During aging, the development of ventricular dysfunction is often
accompanied by a decrease in the capacity of myocytes to respond to
inotropic interventions such as ouabain, -adrenergic stimulation
(1, 21), and -adrenergic stimulation
(19). The present study shows that the response of
Ito to 1-adrenergic stimulation
is similar in aged and young adult myocytes. PE suppressed peak
Ito in both populations by decreasing channel
availability to the same extent. It is well known that the inotropic
effect of 1-adrenergic stimulation is mediated by
multiple mechanisms, including the suppression of
Ito. The present results suggest that
1-adrenoceptor-mediated suppression of
Ito is not involved in the aging-related decline
in inotropic responsiveness to 1-adrenergic stimulation
reported previously (19).
The present study also demonstrates an aging-associated reduction in
ICa,L accompanied by slower inactivation in both
physiological solutions and solutions used to minimize contamination by
other currents. These results are also contradictory to previous
studies that showed no difference in ICa,L in
young and aged myocytes of Wistar rats (30,
32). Again, the discrepancy between the present and
previous studies might be attributable to different experimental
conditions. First, the current density of ICa,L
of myocytes in culture for 48 h is stable and greater than that of freshly isolated myocytes (unpublished data), possibly because of
partial injury in freshly isolated cells after enzyme treatment. Second, previous studies (30, 32) measured
ICa,L under conditions that do not isolate
ICa,L efficiently, e.g., using pipette solutions containing Na+ and a high concentration of Cl
and external solutions containing Na+ and/or
K+. Under these conditions, we also found no significant
difference in peak ICa,L measured at 0 mV in the
two age groups (Fig. 1). The aging-associated change is observed only
when ICa,L is isolated using at least
Na+- and K+-free pipette solutions (Fig. 6). In
dog Purkinje fibers perfused with Na+-free and
Ca2+-rich solutions, slow-response APs, which result
primarily from activation of ICa,L, showed a
decrease in amplitude and overshoot with age (28). In
addition, radioligand binding studies with dihydropyridines in hamster
ventricular membranes showed a decline in binding site density during
senescence (15). A reduction in
ICa,L density was also reported in aged rat
colonic smooth muscle cells (33). As indicated above,
although the magnitude of ICa,L was decreased in
aged myocytes, the slow component of ICa,L
inactivation in aged myocytes was significantly slower than that in
young adult myocytes (Fig. 7). Because of this slow inactivation, the
integrated area of ICa,L in aged myocytes during
the depolarizing voltage pulse was larger than that in young
myocytes. This might account, at least in part, for the prolonged
durations of isometric contraction and the Ca2+ transient
in rat papillary muscle (26, 31) and for the
increased duration of isotonic contraction in rat ventricular myocytes
(10). However, we did not detect a significant increase in
the duration of cell shortening of intact rat ventricular myocytes
(unpublished data), probably because of its short AP.
When young adult myocytes are compared, the present study showing that
APD of aged F344 ventricular myocytes monitored in normal Tyrode
solution was prolonged only at APD90 is different from
those shown in aged myocytes (5, 30) or
papillary muscle (31) of Wistar rats perfused with
solutions containing 2 or 2.5 mM Ca2+. The cause of the
discrepancy is also unclear. High concentrations of Cl in
the pipette solution may have contributed to the low resting membrane
potential observed in previous studies, because a finite Cl conductance could shift membrane potential toward more
positive potentials. In addition, the studies in Wistar rats raise some concerns about species variation compared with results from the present
study using F344 rats. In the present study, the aging-associated increase in Ito and decrease in
ICa,L could shorten APD in aged F344 myocytes.
Both aging-related changes in Ito and
ICa,L are also consistent with the observed
decreased AP amplitude in aged myocytes. However, there is always some
question as to whether papillary muscle represents ventricular myocytes
and whether APs measured using conventional microelectrodes filled with
3 M KCl are comparable to those measured in the whole cell
configuration. Nevertheless, the fact that the AP configuration is
determined by the sum of total activated membrane currents always makes
the interpretation of changes in APD complicated and difficult. Note that the aging-associated changes in the magnitudes of
Ito (>35%) and ICa,L
(~15%) (measured using the step voltage-pulse protocol) do not
parallel observed alterations in APD. The short APD of rat ventricular
myocytes limits the influence of aging-associated changes in time- and
voltage-dependent membrane currents (e.g., both Cd2+- and
4-AP-sensitive currents are completely inactivated in first 10 ms of
the AP). However, previous studies (9, 28)
with other mammalian preparations such as dog Purkinje fibers and human atrial fibers have shown an age-related increase in the initial repolarization (phase 1) of the AP followed by a decrease in plateau magnitude. The increase in phase 1 repolarization has been suggested to
result from an increase in transient outward currents (9); the decreased plateau amplitude is consistent with a reduction of
ICa,L. In addition, if the decreased
ICa,L occurs in all myocytes of aged subjects,
this change could account for many of the reported electrophysiological
changes in the senescent heart of other animal models, including the
1) smaller overshoot of the AP (9,
27); 2) lower plateau height of the AP
(9, 27, 28); 3)
prolongation of AV conduction time or P-R interval (6,
11, 27, 29); 4) low
intrinsic heart rate (17, 27,
29) and sinus nodal rate (8); and
5) slow time to peak shortening (10,
17).
In summary, the increase in Ito and the decrease in ICa,L in aged myocytes could act to decrease the plateau phase and shorten APD. However, APD50-75 (equivalent to the plateau phase of AP in other mammalian ventricular myocytes) does not change significantly (but tends to be shorter) during senescence (20, 28), probably because rat ventricular myocytes have an extremely short APD and little delayed rectifier K+ current. Furthermore, the previously reported (18) aging-associated decline in Na+/K+ pump current in aged myocytes could counterbalance the effects of changes in IK1, Ito, and ICa,L on APD. In contrast, senescence-associated changes in Ito and ICa,L could produce dramatic changes in phase 1 and 2 of the AP in other mammalian myocytes, which have a longer APD than rat myocytes, thereby altering excitation-contraction coupling. These aging-induced changes in cardiac electrophysiology may play important roles in the decreased myocardial function in the elderly.
| |
ACKNOWLEDGEMENTS |
|---|
We thank M.-Y. Liu for excellent technical assistance, Dr. G. R. Wenger for generously supplying the aged L-E rats used in this study, and Dr. J. Stimers's helpful discussion.
| |
FOOTNOTES |
|---|
This work was supported in part by grants from the American Heart Association-Arkansas Affiliate, the American Health Assistance Foundation, and the Office of Naval Research.
Address for reprint requests and other correspondence: S. J. Liu, Dept. of Pharmaceutical Sciences, Univ. of Arkansas for Medical Sciences, 4301 West Markham St., MS 522-3, Little Rock, AR 72205 (E-mail: LiuShi{at}exchange.uams.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.
Received 6 October 1999; accepted in final form 16 February 2000.
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TOP
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RESULTS
DISCUSSION
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3 hearts.
