(Hypertension. 1996;27:265-268.)
© 1996 American Heart Association, Inc.
Articles |
Impaired Regulation of Cell Communication by ß-Adrenergic Receptor Activation in the Failing Heart
From the Departments of Pharmacology and Anesthesiology, School of Medicine, University of Puerto Rico, San Juan.
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Abstract |
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Abstract We investigated the influence of ß-adrenergic receptor activation on the control of gap junctional conductance (gj) in the heart of cardiomyopathic hamsters (11 months old). We measured gj in isolated ventricular cell pairs using two voltage-clamp circuits. Administration of isoproterenol (10-6 mol/L) to the bath had no effect on gj in myopathic cell pairs but increased gj by 45±3% (±SE) in normal hamsters. Moreover, forskolin (10-7 mol/L), an activator of adenyl cyclase, did not change gj in myopathic cells but enhanced gj by 23±2.8% in controls. Similar results were obtained with isobutylmethylxanthine (10-6 mol/L), a phosphodiesterase inhibitor. Dibutyryl-cAMP (10-6 mol/L), however, increased gj of cardiomyopathic cell pairs by 58±2.1% within 2 minutes and enhanced gj in controls by 50±3.6%. The effect of dibutyryl-cAMP on gj of myopathic cells was suppressed by intracellular dialysis of an inhibitor of protein kinase A. These observations indicate that the regulation of gj by the ß-adrenergic receptor–G protein–adenyl cyclase signaling system is greatly impaired in the failing heart but the ability of cAMP to increase gj is still preserved.
Key Words: receptors, adrenergic, beta activation • gap junctions • heart
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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It is well known that the adrenergic receptor–G protein–adenyl cyclase complex is an important signaling system involved in the control of heart rate and contractility.1 2 3 Evidence is available that cardiac failure is accompanied by alterations in autonomic regulation that include a reduced parasympathetic tonus and increased sympathetic control.4 5
However, with the progress of the disease the heart becomes quite
insensitive to sympathetic
stimulation.6 7 8 This is in part
due to downregulation of ß-adrenergic receptors8 but
is also related to a defective coupling of G protein to adenyl
cyclase.9 Indeed, an increase in the 
-subunit of
inhibitory G protein (Gi)
was confirmed by pertussis toxin–catalyzed ADP
ribosylation.10 11 12 Furthermore,
myocardial Gi
mRNA levels are increased in terminal heart
failure.10 13
In cardiomyopathic hamsters the adenyl cyclase
activation in cardiac muscle is clearly reduced in the presence of
fluoride ion, forskolin, isoproterenol, or guanine
nucleotides.9
Previous findings indicate that the activation of ß-adrenergic receptors plays an important role in the regulation of cell-to-cell communication in normal heart muscle.14 15 16 17 Indeed, when isoproterenol is added to the bath the junctional conductance (gj) measured in rat isolated heart cell pairs is increased by 40% within 20 seconds.18 19 Moreover, dibutyryl-cAMP enhances dye coupling in dog muscle trabeculae,20 and the intracellular administration of cAMP increases the electrical coupling of canine Purkinje cells.15 In these studies it was found that the activation of cAMP-dependent protein kinase is involved in the effect of isoproterenol on gj because the intracellular administration of a protein kinase A inhibitor abolished the effect of isoproterenol.18 21
The mechanism by which cAMP increases gj is probably through the phosphorylation of gap junction proteins.14 15 22 23 Other observations have demonstrated that the phosphorylated amino acid involved is serine.22 As stressed before14 the hormonal system is integrated with the intercellular communication system by gap junctions, providing an additional role for hormones: the regulation of the exchange of electrical and chemical signals between cells. The implication of a defect in the adrenergic receptor–G protein–adenyl cyclase signaling system on the regulation of heart cell communication in the failing heart is not known.
In the present work we investigated this important problem in ventricular cell pairs isolated from cardiomyopathic hamsters (11 months old) in which extensive calcification and hypertrophy of the left ventricle have been described.24 25 The TO-2 strain of cardiomyopathic hamster is characterized by several hereditary changes, including ventricular dilation and death by congestive heart failure.24 25
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Male TO-2 cardiomyopathic Syrian hamsters (11 months old) (Biobreeders, Fitchburg, Mass) and healthy male F1B control hamsters of the same age were used. Both the control and cardiomyopathic animals were kept in air-conditioned facilities at the animal house on a normal laboratory animal diet and tap water ad libitum. The animals were anesthetized with sodium pentobarbital (35 mg/kg IP), and the heart was removed with animals under deep anesthesia.
Cell pairs were obtained by enzymatic dispersion of hamster ventricle following the method of Powell and Twist26 and Tanigushi et al.27
The heart was removed and immediately perfused with normal Krebs' solution containing (mmol/L) NaCl 136.5, KCl 5.4, CaCl2 1.8, MgCl2 0.53, NaH2PO4 0.3, NaHCO3 11.9, glucose 5.5, and HEPES 5, with pH adjusted to 7.4. After 20 minutes a calcium-free solution containing collagenase (0.4%) (Worthington Biochemical Corp) was recirculated through the heart for 1 hour. The collagenase solution was washed out with 100 mL of recovery solution containing (mmol/L) taurine 10, oxalic acid 10, glutamic acid 70, KCl 25, KH2PO4 10, glucose 11, and EGTA 0.5, with pH adjusted to 7.4. All solutions were oxygenated with 100% O2.
The ventricles were minced (1 to 2 mm thick slices), and the resulting solution was agitated gently with a Pasteur pipette. The suspension was filtered through nylon gauze and the filtrate centrifuged for 4 minutes at 22g. The cell pellets were then resuspended in normal Krebs' solution. All experiments were made at 36°C.
Suction pipettes were pulled from microhematocrit tubing (Clark
Electromedical Instruments) by means of a controlled puller
(Narishige), and their tips were polished with a microforge
(Narishige). The pipettes, which were prepared immediately before the
experiment, were filled with the following solution (mmol/L): KCl 125,
NaCl 10, MgCl2 3, EGTA 5, and HEPES 10, with pH adjusted to
7.4. The resistance of the filled pipettes (about 2 µm in diameter)
varied from 0.5 to 1.5 M
.
Drugs
Isoproterenol, dibutyryl-cAMP, forskolin,
isobutylmethylxanthine, and the protein kinase
A inhibitor were from Sigma Chemical Co.
Experimental Procedures
All experiments were performed in a
small chamber mounted on the
stage of an inverted phase-contrast microscope (Diaphot, Nikon).
The junctional resistance was determined in cell pairs with the use of
two separated voltage-clamp circuits. Gigaohm sealing was achieved
in each cell, and then the surface cell membrane of both cells was
broken by application of a stronger suction (-30 to -65 cm
H2O) and a whole-cell clamp configuration was produced.
Each pipette was connected to a separated voltage-clamp amplifier
(Dagan Corp) that made possible the control of the nonjunctional
membrane potential in each cell as well as the voltage gradient across
the intercellular junction.
The experimental procedure consisted of holding the membrane potential of both cells at -40 mV. Cell 1 was then pulsed to 0 mV while the membrane potential of cell 2 was maintained unchanged. A voltage was created across the junctional membrane (V1), and a compensating current of opposite polarity recorded from pipette 2 (I2) represents the current flowing through the gap junction. As I2 equals V1/rj, the junctional resistance (rj) can be easily estimated.21 Data acquisition and command potentials were controlled with PCLAMP software (Axon Instruments).
Series resistances
(Rs1 and Rs2) originating
from the tips of the micropipettes were compensated electronically
before the experiment and checked periodically during the experiment.
When necessary, the gj was corrected taking into consideration
the changes in series resistance. For this, the following equation was
used:
gj=
I2/V-(Rs1 I1 Rs2 I2).
Change of the patch electrode solution was made with fine polyethylene tubing. In this way, the protein kinase A inhibitor was introduced into the cell.
Voltage and current signals were displayed simultaneously on an oscilloscope (Tektronix 5113) and chart recorder (Gould 2400). Numerical data are expressed as mean±SE. Student's t test was used to estimate statistical significance, defined at a value of P<.05.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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To investigate the influence of isoproterenol on gj in cell pairs of cardiomyopathic hamsters, we determined gj values under control conditions and then added the ß-adrenergic agonist to the bath and carefully monitored gj. Fig 1B
represents the result from 15
cell pairs and shows that isoproterenol
(10-6 mol/L) caused a negligible increase
of gj. These findings contrast with those obtained in cell pairs
of normal hamsters, in which the same drug concentration elicited an
increment in gj of 45±3% (n=13) (Fig 1A). To
investigate
whether these changes in gj were related to an increase in
surface cell membrane resistance, we measured the time constant (tm) of
the cell membrane using electrotonic potentials (Fig 2)
recorded from single cells under the current clamp
configuration.
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The rationale here is as follows: tm=RmCm, where Rm is the membrane resistance and Cm is the membrane capacitance, which is constant. Therefore, changes in Rm lead to variations of tm. Measurements of tm were performed under control conditions and after isoproterenol administration. Results from eight experiments indicated that the average tm at control conditions (20±2.8 milliseconds) was not statistically different from that recorded after isoproterenol (10-6 mol/L) administration (18.9±4 milliseconds) (P>.05). Moreover, no change in series resistance was found during these experiments.
To investigate whether the lack of action of isoproterenol on gj
was solely due to downregulation of ß-adrenergic receptors, we
used forskolin, an activator of adenyl cyclase. Fig 3 shows
results from a single experiment with a normal
(A) and myopathic (B) cell pair. Experiments performed on several cell
pairs indicated that forskolin (10-7
mol/L) added to the bath increased gj by 23±2.8% (n=16) within
2.5 minutes in normal hamster (Fig 4B). In myopathic
cell pairs, however, forskolin (10-7
mol/L) was unable to change gj in 12 experiments but elicited a
negligible increase (0.93%) of gj in six additional cell pairs
(see Fig 4). The effect of forskolin on gj of cell pairs
isolated from the ventricle of normal hamsters was not related to a
change in surface cell membrane resistance or sealing resistance.
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To investigate further the influence of the cAMP cascade on gj
of myopathic cell pairs, we used
isobutylmethylxanthine, a phosphodiesterase
inhibitor. When the compound
(10-6 mol/L) was added to the bath, the
gj was not significantly altered (see Fig 5).
These findings contrast with those obtained in normal hamsters, in
which an increment of 38±1.5% (n=12) was found after the
addition of
isobutylmethylxanthine (see Fig 5, bottom).
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The question remains whether cAMP is able to increase gj in
myopathic cells, as described in other cardiac
preparations.15 To investigate this point we added
dibutyryl-cAMP (10-6 mol/L), which
crosses the cell membrane easily, to the bath and carefully monitored
changes in gj. As can be seen in Fig 6A, which is
the result from a single myopathic cell pair, dibutyryl-cAMP
increased gj by 57%. The increment in gj started in
about 25 seconds and reached a maximal and steady level within 2
minutes. Experiments performed on 14 myopathic cell pairs showed an
average increase of gj of 58±2.1% (Fig 7,
left), whereas in normal hamsters the same concentration
of dibutyryl-cAMP caused an increment of 50±3.6% in
gj.
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The increment in gj elicited by dibutyryl-cAMP in myopathic cell pairs is not related to a change in surface cell membrane resistance, as judged by the lack of action of the compound on tm. Indeed, measurements of tm made on eight single myopathic cells indicated an average value of 21±3 milliseconds before and 20.7±2.1 milliseconds 3 minutes after dibutyryl-cAMP (10-6 mol/L) administration. In addition, no change in series resistance was found during these experiments.
To investigate whether the effect of dibutyryl-cAMP on gj
was related to activation of cAMP-dependent protein kinase, we added an
inhibitor of this kinase (Walsh inhibitor) (20
µg/mL) to the pipette solution and dialyzed the compound into the
cell for 4 minutes. After this time dibutyryl-cAMP
(10-6 mol/L) was added to the bath, and
changes in gj were monitored. The kinase inhibitor
by itself caused no significant change in gj. However, as shown
in Fig 7
, right, the kinase inhibitor suppressed the effect
of dibutyryl-cAMP on gj (average from seven
experiments).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present results indicate that gj regulation by a ß-adrenergic agonist in the ventricle of the failing heart is severely impaired. Since forskolin, an activator of adenyl cyclase, was also unable to increase gj in myopathic cell pairs, it is possible to conclude that the fault involves steps distal to the receptors themselves.
It is known that there are G protein abnormalities in the cardiac muscle of myopathic hamsters as well as in human heart failure.9 28 It is important to point out that these abnormalities were not found in young cardiomyopathic hamsters but appeared in 100-day-old animals in which the cardiomyopathy was clearly advanced. Although no information is available on much older hamsters like those used in the present studies, it is reasonable to think that these alterations in the ß-adrenergic receptor–G protein–adenyl cyclase signaling system are of the same or even larger magnitude than those described in 100-day-old animals.
Our present results seem to indicate that the downregulation of ß-receptors and the defect in ß-receptor–adenyl cyclase coupling are both responsible for the lack of action of isoproterenol on gj. Age by itself seems to have no influence on these results because in normal hamsters of the same age (11 months old) both isoproterenol and forskolin were able to increase gj. The extremely small effect of phosphodiesterase inhibition on gj of myopathic cell pairs is probably due to a reduced activity of adenyl cyclase, which led to a decreased formation of cAMP despite the inhibition of phosphodiesterase. Indeed, evidence is available that the phosphodiesterase activity is not altered in human heart failure.29
The increment in gj caused by dibutyryl-cAMP in myopathic cell pairs and the abolishment of this effect by the protein kinase A inhibitor indicate that the ability of cAMP to activate protein kinase A with a consequent increase in gj is still preserved in these animals. This finding can be of strategic value not only for the control of cell communication (and possibly cardiac arrhythmias) but also for the increment of heart contractility. Both effects might be of potential help to patients with congestive heart failure.
The present results indicate that sympathetic stimuli are unable to increase gj in myopathic cell pairs. This means that the enhanced conduction velocity (which depends on gj) usually seen under ß-adrenergic activation is not present in the failing heart at an advanced stage of the disease. Furthermore, the increases in gj and consequently in the electrical synchronization that occurs in the normal heart when sympathetic nerve endings are depolarized or epinephrine is released into the bloodstream are not present in the failing heart. This creates a serious impairment of the autonomic regulation of the failing cardiac muscle, thereby preventing cardiovascular adjustments that are essential for cardiovascular homeostasis.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (HL-34148, 532943, and RR-03051) and Merck Sharp & Dohme.
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Footnotes |
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Reprint requests to Dr Walmor C. De Mello, Department of Pharmacology, School of Medicine, University of Puerto Rico, PO Box 365067, San Juan, PR 00936-5067.
Received September 25, 1995; first decision October 23, 1995; accepted October 23, 1995.
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